Osteogenic devices and methods of use thereof for repair of endochondral bone and osteochondral defects

ABSTRACT

Disclosed herein are improved osteogenic devices and methods of use thereof for repair of bone and cartilage defects. The devices and methods promote accelerated formation of repair tissue with enhanced stability using less osteogenic protein than devices in the art. Defects susceptible to repair with the instant invention include, but are not limited to: critical size defects, non-critical size defects, non-union fractures, fractures, osteochondral defects, subchondral defects, and defects resulting from degenerative diseases such as osteochondritis dessicans.

FIELD OF THE INVENTION

The invention disclosed herein relates to materials and methods forrepairing bone and cartilage defects using osteogenic proteins.

BACKGROUND OF THE INVENTION

A class of proteins now has been identified that is competent to act astrue chondrogenic tissue morphogens. That is, these proteins are able,on their own, to induce the proliferation and differentiation ofprogenitor cells into functional bone, cartilage, tendon, and/orligamentous tissue. This class of proteins, referred to herein as“osteogenic proteins” or “morphogenic proteins” or “morphogens,”includes members of the family of bone morphogenetic proteins (BMPs)which were initially identified by their ability to induce ectopic,endochondral bone morphogenesis. The osteogenic proteins generally areclassified in the art as a subgroup of the TGF-β superfamily of growthfactors (Hogan (1996) Genes & Development 10:1580–1594). Members of themorphogen family of proteins include the mammalian osteogenic protein-1(OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenicprotein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3),BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP),BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and itsmurine homolog Vgr-1, BMP-9, BMP-10, BMP11, BMP-12, GDF3 (also known asVgr2), GDF8, GDF9, GDF10, GDF11, GDF12, BMP-13, BMP-14, BMP-15, GDF-5(also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7(also known as CDMP-3), the Xenopus homolog Vg1 and NODAL, UNIVIN,SCREW, ADMP, and NEURAL. Members of this family encode secretedpolypeptide chains sharing common structural features, includingprocessing from a precursor “pro-form” to yield a mature polypeptidechain competent to dimerize and containing a carboxy terminal activedomain, of approximately 97–106 amino acids. All members share aconserved pattern of cysteines in this domain and the active form ofthese proteins can be either a disulfide-bonded homodimer of a singlefamily member or a heterodimer of two different members (see, e.g.,Massague (1990) Annu. Rev. Cell Biol. 6:597; Sampath, et al. (1990) J.Biol. Chem. 265:13198). See also, U.S. Pat. No. 5,011,691; U.S. Pat. No.5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085–2093, Wharton et al.(1991) PNAS 88:9214–9218), (Ozkaynak (1992) J. Biol. Chem.267:25220–25227 and U.S. Pat. No. 5,266,683); (Celeste et al. (1991)PNAS 87:9843–9847); (Lyons et al. (1989) PNAS 86:4554–4558). Thesedisclosures describe the amino acid and DNA sequences, as well as thechemical and physical characteristics, of these osteogenic proteins. Seealso, Wozney et al. (1988) Science 242:1528–1534); BMP 9 (WO93/00432,published Jan. 7, 1993); DPP (Padgett et al. (1987) Nature 325:81–84;and Vg-1 (Weeks (1987) Cell 51:861–867).

Thus true osteogenic proteins capable of inducing the above-describedcascade of morphogenic events resulting in endochondral bone formation,have now been identified, isolated, and cloned. Whethernaturally-occurring or synthetically prepared, these osteogenic factors,when implanted in a mammal in association with a matrix or substratethat allows attachment, proliferation and differentiation of migratoryprogenitor cells, can induce recruitment of accessible progenitor cellsand stimulate their proliferation, thereby inducing differentiation intochondrocytes and osteoblasts, and further inducing differentiation ofintermediate cartilage, vascularization, bone formation, remodeling,and, finally, marrow differentiation. Furthermore, numerouspractitioners have demonstrated the ability of these osteogenicproteins, when admixed with either naturally-sourced matrix materialssuch as collagen or synthetically-prepared polymeric matrix materials,to induce bone formation, including endochondral bone formation, underconditions where true replacement bone otherwise would not occur. Forexample, when combined with a matrix material, these osteogenic proteinsinduce formation of new bone in Large segmental bone defects, spinalfusions, and fractures.

Naturally-sourced matrices, such as collagen, can be replaced with inertmaterials such as plastic, but plastic is not a suitable substitutesince it does not resorb and is limited to applications requiring simplegeometric configurations. To date, biodegradable polymers and copolymershave also been used as matrices admixed with osteogenic proteins forrepair of non-union defects. While such matrices may overcome some ofthe above-described insufficiencies, use of these matrices necessitatesdetermination and control of features such as polymer chemistry,particle size, biocompatability and other particulars critical foroperability. For example, pores must be formed in the polymer in amanner which ensures adsorption of protein into the matrix andbiodegradation of the matrix. Prior to use of the polymeric matrix,therefore, it is necessary to undergo the extra step of treating thepolymer to induce the formation of pores of the appropriate size.

Standard osteogenic devices, which include either collagen or polymermatrices in admixture with osteogenic protein, lend themselves lessamenable to manipulation during surgery. Standard osteogenic devicesoften have a dry, sandy consistency and can be washed away whenever thedefect site is irrigated during surgery, and/or by blood and/or otherfluids infiltrating the site post-surgery. The addition of certainmaterials to these compositions can aid in providing a more manageablecomposition for handling during surgery. U.S. Pat. Nos. 5,385,887;5,520,923; 5,597,897 and International Publication WO 95/24210 describecompositions containing a synthetic polymer matrix, osteogenic protein,and a carrier for such a purpose. Such compositions have been limited,however, to synthetic polymer matrices because of a desire to overcomecertain alleged adverse immunologic reactions contemplated associatedwith other types of matrices especially biologically-derived matrices,including some forms of collagen. These compositions, therefore, sufferfrom the same feasibility concerns for optimizing polymer chemistry,particle size, biocompatability, etc., described above.

Needs remain for compositions and methods for repairing bone andcartilage defects which provide greater ease in handling during surgeryand which do not rely on synthetic polymer matrices. Needs also remainfor methods and compositions that can enhance the rate and quality ofnew bone and cartilage formation.

Accordingly, it is an object of the instant invention to provideimproved osteogenic devices and methods of use thereof for repairingbone defects, cartilage defects and/or osteochondral defects that: areeasier to manipulate during surgery; circumvent the concerns of polymerchemistry, particle size and biocompatibility associated with the use ofsynthetic polymer matrices; and, which permit accelerated bone formationand more stable cartilage repair using lower doses of osteogenic proteinthan can be achieved using devices and methods now in the art. It is afurther object of the instant invention to provide osteogenic devicesand methods of use thereof for repairing non-healing, non-union defectsand for promoting articular cartilage repair in chondral orosteochondral defects. Yet another object of the instant invention is toprovide devices and methods for repair of bone and cartilage defectswithout surgical intervention. These and other objects, along withadvantages and features of the invention disclosed herein, will beapparent from the description, drawings and claims that follow.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that admixing osteogenicprotein and a non-synthetic, non-polymeric matrix such as collagen witha binding agent yields an improved osteogenic device with enhanced boneand cartilage repair capabilities. Not only can such improved devicesaccelerate the rate of repair, these devices also can promote formationof high quality, stable repair tissue, particularly cartilage tissue.Additionally, the foregoing benefits can be achieved using significantlyless osteogenic protein than required by standard osteogenic devices.While not wishing to be bound by theory, the aforementioned unexpectedproperties likely can be attributed to a complementary or synergisticinteraction between the non-polymeric matrix and the binding agent. Inview of existing orthopedic and reconstructive technologies, thesediscoveries are unexpected and were heretofore unappreciated.

The invention provides, in one aspect, a novel device for inducing localbone and cartilage formation comprising osteogenic protein, matrixderived from non-synthetic, non-polymeric material, and binding agent.As contemplated herein, the device preferably comprises osteogenicproteins such as, but not limited to OP-1, OP-2, BMP-2, BMP-4, BMP-5 andBMP-6. A currently preferred osteogenic protein is OP-1. As used herein,the terms “morphogen”, “bone morphogen”, “bone morphogenic protein”,“BMP”, “osteogenic protein” and “osteogenic factor” embrace the class ofproteins typified by human osteogenic protein 1 (hOP-1). Nucleotide andamino acid sequences for hOP-1 are provided in Seq. ID Nos. 1 and 2,respectively. For ease of description, hOP-1 is recited herein below asa representative osteogenic protein. It will be appreciated by theartisan of ordinary skill in the art, however, that OP-1 merely isrepresentative of the TGF-β subclass of true tissue morphogens competentto act as osteogenic proteins, and is not intended to limit thedescription. Other known, and useful proteins include, BMP-2, BMP-3,BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,GDF-10, GDF-11, GDF-12, NODAL, UNIVIN, SCREW, ADMP, NEURAL andosteogenically active amino acid variants thereof. In one preferredembodiment, the proteins useful in the invention include biologicallyactive species variants of any of these proteins, including conservativeamino acid sequence variants, proteins encoded by degenerate nucleotidesequence variants, and osteogenically active proteins sharing theconserved seven cysteine skeleton as defined herein and encoded by a DNAsequence competent to hybridize to a DNA sequence encoding an osteogenicprotein disclosed herein, including, without limitation, OP-1, BMP-5,BMP-6, BMP-2, BMP-4 or GDF-5, GDF-6 or GDF-7. In another embodiment,useful osteogenic proteins include those sharing the conserved sevencysteine domain and sharing at least 70% amino acid sequence homology(similarity) within the C-terminal active domain, as defined herein. Instill another embodiment, the osteogenic proteins of the invention canbe defined as osteogenically active proteins having any one of thegeneric sequences defined herein, including OPX (SEQ ID No: 3) andGeneric Sequences 7 and 8, or Generic Sequences 9 and 10.

OPX accommodates the homologies between the various species of theosteogenic. OP1 and OP2 proteins, and is described by the amino acidsequence presented herein below and in SEQ ID NO: 3. Generic sequence 9is a 96 amino acid sequence containing the six cysteine skeleton definedby hOP1 (residues 335–431 of SEQ ID NO: 2) and wherein the remainingresidues accommodate the homologies of OP1, OP2, OP3, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1, GDF-3,GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, UNIVIN, NODAL,DORSALIN, NURAL, SCREW and ADMP. That is, each of the non-cysteineresidues is independently selected from the corresponding residue inthis recited group of proteins. Generic Sequence 10 is a 102 amino acidsequence which includes a 5 amino acid sequence added to the N-terminusof the Generic Sequence 9 and defines the seven cysteine skeleton ofhOP1 (330–431 SEQ ID NO: 2). Generic Sequences 7 and 8 are 96 and 102amino acid sequences, respectively, containing either the six cysteineskeleton (Generic Sequence 7) or the seven cysteine skeleton (GenericSequence 8) defined by hOP1 and wherein the remaining residuesnon-cysteine accommodate the homologies of: OP-1, OP-2, OP-3, BMP2,BMP3, BMP4, 60A, DPP, Vg1, BMP5, BMP6, Vgr-1, and GDF-1.

As taught below, preferred matrices are non-synthetic, non-polymericmaterials and can be naturally-sourced or derived from biologicalmaterials. Examples of preferred matrices include, but are not limitedto, collagen and demineralized bone. One currently preferred matrix iscollagen. Thus, the devices of the instant invention do not comprise asa primary component synthetic polymeric matrices such as homopolymers orcopolymers of α-hydroxy acetic acid and/or α-hydroxy propionic acid,including racemic mixtures thereof.

With respect to binding agents, the instant devices preferably compriseagents useful as viscosity-increasing, suspending and/or emulsifyingagents. In particular, cellulosic derivatives are preferred. A currentlypreferred group of binding agents is the alkycellulose group; especiallymethylcelluloses such as carboxymethylcellulose. Other suitable bindingagents include other cellulose gums, sodium alginate, dextrans andgelatin powder. In certain embodiments, the improved devices of theinstant invention further comprise a wetting agent such as, but notlimited to, saline or other aqueous physiological solution.

The improved devices of the instant invention can assume a variety ofconfigurations. The configuration will depend, in part, upon the type ofbinding agent and wetting agent employed. As disclosed herein, onecurrently preferred embodiment can have a putty consistency. Thisparticular configuration is especially suitable for treating opendefects in accordance with the methods of the instant invention. Anothercurrently preferred embodiment of improved osteogenic device can have aviscous fluid consistency. This particular configuration is especiallysuitable for treating closed defects in accordance with the methodsdisclosed herein. Depending upon the configuration of the improveddevice, providing it to a defect site can be accomplished by a varietyof delivery modes. For example, a putty can be packed in and/or aroundthe defect or extruded as a bead from a large-bore apparatus.Alternatively, a viscous liquid can be injected into and/or around thedefect, or alternatively brushed and/or painted on the defect'ssurface(s). Exploitation of a variety of these possible embodiments torepair bone and cartilage defects is exemplified herein.

Among the characteristics of a preferred binding agent is an ability torender the device: pliable, shapeable and/or malleable; injectable;adherent to bone, cartilage, muscle and other tissues; resistant todisintegration upon washing and/or irrigating during surgery; and,resistant to dislodging during surgery, suturing and post-operatively,to name but a few. Additionally, in certain preferred embodiments, abinding agent can achieve the aforementioned features and benefits whenpresent in low proportions. For example, a currently preferred improveddevice comprises approximately 1 part binding agent and approximately 5parts matrix. Another currently preferred device comprises approximately3 parts binding agent to 5 parts matrix. Certain binding agents can beused in equal or greater proportions relative to matrix. Anothercurrently preferred device comprises 1 part binding agent and 3 partsmatrix. As exemplified herein, improved devices of widely divergentproportions can induce bone and cartilage formation. Exemplified hereinare improved devices having parts of binding agent to parts of matrixranging from approximately 1:1 to 4:1 as well as from approximately 1:2to 1:5. Any proportion of binding agent to matrix can be used topractice the instant invention.

Furthermore, the instant invention contemplates that an improvedosteogenic device can comprise more than one matrix material incombination; the relative proportions can be varied to achieve thedesired clinical outcome and can be routinely determined using ordinaryskill. A currently preferred matrix is collagen, especially bovinecollagen. Another suitable matrix is demineralized bone. Yet othersuitable matrices are hydroxyapatites (HAp) of varying calcium:phosphate (Ca/P) molar ratios, porosity and crystallinity; bioactiveceramics; and calcium phosphate ceramics, to name but a few.Additionally, admixtures of the foregoing whereinHAp/tricalciumphosphate ratios are manipulated are also contemplatedherein.

In another aspect, the instant invention provides methods for inducinglocal bone or cartilage formation for repair of bone, cartilage orosteochondral defects. The instant methods are contemplated as useful toinduce formation of at least endochondral bone, intramembranous bone,and articular cartilage. As disclosed herein, methods of repair includetreatment of both closed and open defects with the above-describedimproved osteogenic devices. As taught herein, the methods of theinstant invention can be practiced using improved devices that are ofsufficient volume to fill the defect site, as well as using improveddevices that are not. Moreover, as a result of this discovery,embodiments are now available for promoting bone and/or cartilage defectrepair without requiring surgical intervention. Availability of suchmethods has implications for compromised individuals such as diabetics,smokers, obese individuals and others whose overall health and impairedblood flow to their extremities are placed at risk when surgicalintervention is required. Examples of defects include, but are notlimited to, critical size defects, non-critical size defects, non-unionfractures, fractures, osteochondral defects, chondral defects andperiodontal defects.

In another aspect, the instant invention provides a kit for practice ofthe above-described methods. As contemplated herein, one embodiment of akit for inducing local bone formation or cartilage formation comprisesan improved device wherein the osteogenic protein and matrix arepackaged in the same receptacle. In other embodiments, the osteogenicprotein, matrix and binding agent are in the same receptacle. In yetother embodiments, wetting agent is also provided and packagedseparately from the other kit components.

Because the instant invention provides practitioners with improvedmaterials and methods for bone and cartilage repair, including repair ofarticular cartilage present in mammalian joints, it overcomes problemsotherwise encountered using the methods and devices of the art. Forexample, the instant invention can induce formation of bona fide hyalinecartilage rather than fibrocartilage at a defect site. Functionalhyaline cartilage forms on the articulating surface of bone at a defectsite and does not degenerate over time to fibrocartilage. By contrast,prior art methods generally ultimately result in development offibrocartilage at the defect site. Unlike hyaline cartilage,fibrocartilage lacks the physiological ability to restore articulatingjoints to their full capacity. Thus, when improved osteogenic devicesare used in accordance with the instant methods, the practitioner cansubstantially restore an osteochondral or a chondral defect in afunctionally articulating joint and avoid the undesirable formation offibrocartilage typical of prior art methods. As contemplated herein, theinvention further embodies allogenic replacement materials for repairingavascular tissue in a skeletal joint which results in formation ofmechanically and functionally viable replacement tissues at a joint.

In summary, the methods, devices, and kits of the present invention canbe used to induce endochondral or intramembranous bone formation forrepairing bone defects which do not heal spontaneously, as well as forpromoting and enhancing the rate and/or quality of new bone formation,particularly in the repair of fractures and fusions, including spinalfusions. The methods, devices, and kits also can induce repair ofosteochondral and/or subchondral defects, i.e., can induce formation ofnew bone and/or the overlying surface cartilage. The present inventionis particularly suitable for use in repair of defects resulting fromdeteriorative or degenerative diseases such as, but not limited to,osteochondritis dessicans. It is also particularly suitable for use inpatients requiring repetitive reconstructive surgeries, as well ascancer patients. Other applications include, but are not limited to,prosthetic repair, spinal fusion, scoliosis, cranial/facial repair, andmassive allograft repair.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and features of the invention, as wellas the invention itself, may be more fully understood from the followingdescription, when read together with the accompanying drawings, inwhich:

FIG. 1 is a graph depicting cohesiveness properties of varying parts(w/w) of binding agent to parts (w/w) of standard OP device.

FIG. 2 is a graph depicting the effect of varying volumes of wettingagent on the integrity of an improved osteogenic device.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In order to more clearly and concisely describe the subject matter ofthe claimed invention, the following definitions are intended to provideguidance as to the meaning of specific terms used in the followingwritten description and appended claims.

“Bone formation” means formation of endochondral bone or formation ofintramembranous bone. In humans, bone formation begins during the first6–8 weeks of fetal development. Progenitor stem cells of mesenchymalorigin migrate to predetermined sites, where they either: (a) condense,proliferate, and differentiate into bone-forming cells (osteoblasts), aprocess observed in the skull and referred to as “intramembranous boneformation;” or, (b) condense, proliferate and differentiate intocartilage-forming cells (chondroblasts) as intermediates, which aresubsequently replaced with bone-forming cells. More specifically,mesenchymal stem cells differentiate into chondrocytes. The chondrocytesthen become calcified, undergo hypertrophy and are replaced by newlyformed bone made by differentiated osteoblasts, which now are present atthe site. Subsequently, the mineralized bone is extensively remodeled,thereafter becoming occupied by an ossicle filled with functionalbone-marrow elements. This process is observed in long bones andreferred to as “endochondral bone formation.” In postfetal life, bonehas the capacity to repair itself upon injury by mimicking the cellularprocess of embryonic endochondral bone development. That is, mesenchymalprogenitor stem cells from the bone-marrow, periosteum, and muscle canbe induced to migrate to the defect site and begin the cascade of eventsdescribed above. There, they accumulate, proliferate, and differentiateinto cartilage, which is subsequently replaced with newly formed bone.

“Bone” refers to a calcified (mineralized) connective tissue primarilycomprising a composite of deposited calcium and phosphate in the form ofhydroxyapatite, collagen (primarily Type I collagen) and bone cells suchas osteoblasts, osteocytes and osteoclasts, as well as to bone marrowtissue which forms in the interior of true endochondral bone. Bonetissue differs significantly from other tissues, including cartilagetissue. Specifically, bone tissue is vascularized tissue composed ofcells and a biphasic medium comprising a mineralized, inorganiccomponent (primarily hydroxyapatite crystals) and an organic component(primarily of Type I collagen). Glycosaminoglycans constitute less than2% of this organic component and less than 1% of the biphasic mediumitself, or of bone tissue per se. Moreover, relative to cartilagetissue, the collagen present in bone tissue exists in a highly-organizedparallel arrangement. Bony defects, whether from degenerative, traumaticor cancerous etiologies, pose a formidable challenge to thereconstructive surgeon. Particularly difficult is reconstruction orrepair of skeletal parts that comprise part of a multi-tissue complex,such as occurs in mammalian joints.

“Cartilage formation” means formation of connective tissue containingchondrocytes embedded in an extracellular network comprising fibrils ofcollagen (predominantly Type II collagen along with other minor typessuch as Types IX and XI), various proteoglycans, other proteins andwater. “Articular cartilage” refers specifically to hyaline or articularcartilage, an avascular non-mineralized tissue which covers thearticulating surfaces of the portions of bones in joints and allowsmovement in joints without direct bone-to-bone contact, therebypreventing wearing down and damage of opposing bone surfaces. Normalhealthy articular cartilage is referred to as “hyaline,” i.e. having acharacteristic frosted glass appearance. Under physiological conditions,articular cartilage tissue rests on the underlying, mineralized bonesurface called subchondral bone, which contains highly vascularizedossicles. The articular, or hyaline cartilage, found at the end ofarticulating bones is a specialized, histologically distinct tissue andis responsible for the distribution of load resistance to compressiveforces, and the smooth gliding that is part of joint function. Articularcartilage has little or no self-regenerative properties. Thus, if thearticular cartilage is torn or worn down in thickness or is otherwisedamaged as a function of time, disease or trauma, its ability to protectthe underlying bone surface is comprised. In normal articular cartilage,a balance exists between synthesis and destruction of theabove-described extracellular network. However, in tissue subjected torepeated trauma, for example, due to friction between misaligned bonesin contact with one another, or in joint diseases characterized by netloss of articular cartilage, e.g., osteoarthritis, an imbalance occursbetween synthesis and degradation.

Other types of cartilage in skeletal joints include fibrocartilage andelastic cartilage. Secondary cartilaginous joints are formed by discs offibrocartilage that join vertebrae in the vertebral column. Infibrocartilage, the mucopolysaccharide network is interlaced withprominent collagen bundles and the chondrocytes are more widelyscattered than in hyaline cartilage. Elastic cartilage contains collagenfibers that are histologically similar to elastin fibers. Cartilagetissue, including articular cartilage, unlike other connective tissues,lacks blood vessels, nerves, lymphatics and basement membrane. Cartilageis composed of chondrocytes, which synthesize an abundant extracellularmilieu composed of water, collagens, proteoglycans and noncollagenousproteins and lipids. Collagen serves to trap proteoglycans and toprovide tensile strength to the tissue. Type II collagen is thepredominant collagen in cartilage tissue. The proteoglycans are composedof a variable number of glycosaminoglycan chains, keratin sulphate,chondroitin sulphate and/or dermatan sulphate, and N-lined and O-linkedoligosaccharides covalently bound to a protein core.

Articular, or hyaline, cartilage can be distinguished from other formsof cartilage by both its morphology and its biochemistry. Certaincollagens such as the fibrotic cartilaginous tissues, which occur inscar tissue, for example, are keloid and typical of scar-type tissue,i.e., composed of capillaries and abundant, irregular, disorganizedbundles of Type I and Type II collagen. In contrast, articular cartilageis morphologically characterized by superficial versus mid versus deepzones which show a characteristic gradation of features from the surfaceof the tissue to the base of the tissue adjacent to the bone. In thesuperficial zone, for example, chondrocytes are flattened and lieparallel to the surface embedded in an extracellular network thatcontains tangentially arranged collagen and few proteoglycans. In themid zone, chondrocytes are spherical and surrounded by an extracellularnetwork rich in proteoglycans and obliquely organized collagen fibers.In the deep zone, close to the bone, the collage fibers are verticallyoriented. The keratin sulphate rich proteoglycans increase inconcentration with increasing distance from the cartilage surface. For adetailed description of articular cartilage micro-structure, see, forexample, (Aydelotte and Kuettner, (1988), Conn. Tiss. Res. 18:205;Zanetti et al., (1985), J. Cell Biol. 101:53; and Poole et al., (1984),J. Anat. 138:13. Biochemically, articular collagen can be identified bythe presence of Type II and Type IX collagen, as well as by the presenceof well-characterized proteoglycans, and by the absence of Type Xcollagen, which is associated with endochondral bone formation.

Two types of defects are recognized in articular surfaces, i.e.,full-thickness defects and superficial defects. These defects differ notonly in the extent of physical damage to the cartilage, but also in thenature of the repair response each type of lesion can elicit.Full-thickness defects, also referred to herein as “osteochondraldefects,” of an articulating surface include damage to the hyalinecartilage, the calcified cartilage layer and the subchondral bone tissuewith its blood vessels and bone marrow. Full-thickness defects can causesevere pain, since the bone plate contains sensory nerve endings. Suchdefects generally arise from severe trauma and/or during the late stagesof degenerative joint disease, such a osteoarthritis. Full-thicknessdefects may, on occasion, lead to bleeding and the induction of a repairreaction from the subchondral bone. In such instances, however, therepair tissue formed is a vascularized fibrous type of cartilage withinsufficient biomechanical properties, and does not persist on along-term basis. In contrast, superficial defects in the articularcartilage tissue are restricted to the cartilage tissue itself. Suchdefects, also referred to herein as “chondral” or “subchondral defects”,are notorious because they do not heal and show no propensity for repairreactions. Superficial defects may appear as fissures, divots, or cleftsin the surface of the cartilage. They contain no bleeding vessels (bloodspots), such as those seen in full-thickness defects. Superficialdefects may have no known cause, but they are often the result ofmechanical derangements that lead to a wearing down of the cartilaginoustissue. Such mechanical derangements may be caused by trauma to thejoint, e.g., a displacement of torn meniscus tissue into the joint,meniscectomy, a Taxation of the joint by a torn ligament, malalignmentof joints, or bone fracture, or by hereditary diseases. Superficialdefects are also characteristic of early stages of degenerative jointdiseases, such as osteoarthritis. Since the cartilage tissue is notinnervated or vascularized, superficial defects do not heal and oftendegenerate into full-thickness defects.

“Defect” or “defect site”, as contemplated herein, can define a bonystructural disruption requiring repair. The defect further can define anosteochondral defect, including a structural disruption of both the boneand overlying cartilage. A defect can assume the configuration of a“void”, which is understood to mean a three-dimensional defect such as,for example, a gap, cavity, hole or other substantial disruption in thestructural integrity of a bone or joint. A defect can be the result ofaccident, disease, surgical manipulation, and/or prosthetic failure. Incertain embodiments, the defect is a void having a volume incapable ofendogenous or spontaneous repair. Such defects are generally twice thediameter of the subject bone and are also called “critical size”defects. For example, in a canine ulna defect model, the art recognizessuch defects to be approximately 3–4 cm, generally at leastapproximately 2.5 cm, gap incapable of spontaneous repair. See, forexample, Schmitz et al., Clinical Orthopaedics and Related Research205:299–308 (1986); and Vukicevic et al., in Advanced in Molecular andCell Biology, Vol. 6, pp. 207–224 (1993) (JAI Press, Inc.), thedisclosures of which are incorporated by reference herein. In rabbit andmonkey segmental defect models, the gap is approximately 1.5 cm and 2.0cm, respectively. In other embodiments, the defect is a non-criticalsize segmental defect. Generally, these are capable of some spontaneousrepair, albeit biomechanically inferior to those made possible bypractice of the instant innovation. In certain other embodiments, thedefect is an osteochondral defect, such as an osteochondral plug. Such adefect traverses the entirety of the overlying cartilage and enters, atleast in part, the underlying bony structure. In contrast, a chondral orsubchondral defect traverses the overlying cartilage, in part or inwhole, respectively, but does not involve the underlying bone. Otherdefects susceptible to repair using the instant invention include, butare not limited to, non-union fractures; bone cavities; tumor resection;fresh fractures (distracted or undistracted); cranial/facialabnormalities; periodontal defects and irregularities; spinal fusions;as well as those defects resulting from diseases such as cancer,arthritis, including osteoarthritis, and other bone degenerativedisorders such as osteochondritis dessicans.

“Repair” is intended to mean new bone and/or cartilage formation whichis sufficient to at least partially fill the void or structuraldiscontinuity at the defect. Repair does not, however, mean, orotherwise necessitate, a process of complete healing or a treatmentwhich is 100% effective at restoring a defect to its pre-defectphysiological/structural/mechanical state.

“Matrix”, as contemplated herein, means a non-polymeric, non-syntheticmaterial that can act as an osteoconductive substrate and has ascaffolding structure on which infiltrating cells can attach,proliferate and participate in the morphogenic process culminating inbone formation. As contemplated herein, matrix does not includepolymeric, synthetic materials such as polymeric matrices comprisinghomopolymers or copolymers of α-hydroxy acetic acid and/or α-hydroxyproponic acid, including racemic mixtures thereof. Specifically,matrices as contemplated herein do not include homopolymers orcopolymers of glycolic acid, lactic acid, and butyric acid, includingderivatives thereof. For example, the matrix of the instant inventioncan be derived from biological, or naturally-sourced, ornaturally-occurring materials. A suitable matrix must be particulate andporous, with porosity being a feature critical to its effectiveness ininducing bone formation, particularly endochondral bone formation. It isunderstood that the term “matrix” means a structural component orsubstrate intrinsically having a three-dimensional form upon whichcertain cellular events involved in endochondral bone morphogenesis willoccur; a matrix acts as a temporary scaffolding structure forinfiltrating cells having interstices for attachment, proliferation anddifferentiation of such cells. The instant invention contemplates thatan improved osteogenic device can comprise more than one matrix materialin combination; the relative proportions can be varied to achieve thedesired clinical outcome and can be routinely determined using ordinaryskill. A currently preferred matrix is collagen, especially bovinecollagen. Another suitable matrix is demineralized bone. Yet othersuitable matrices are hydroxyapatites (HAp) of varying calcium:phosphate (Ca/P) molar ratios, porosity and crystallinity; bioactiveceramics; and calcium phosphate ceramics, to name but a few.Additionally, admixtures of the foregoing whereinHAp/tricalciumphosphate ratios are manipulated are also contemplatedherein. These matrices can be obtained commercially in the form ofgranules, blocks and powders. For example, Pyrost® is a HAp blockderived from bovine bone (Osteo AG, Switzerland); Collapt® is a HApsponge containing collagen (Osteo AG, Switzerland); tricalciumphosphates (β-TCP) can be obtained from Pharma GmbH (Germany) asCerasob®; TCP/HAp granule admixtures can be obtained from Osteonics(Netherlands); and 100% HAp powder or granules can be obtained from CAM(a subsidiary of Osteotech, N.J.). Preparation and characterization ofcertain of the aforementioned matrices have been extensively describedin the art and necessitates no more than routine experimentation andordinary skill. See, for example, U.S. Pat. No. 4,975,526; U.S. Pat. No.5,011,691; U.S. Pat. No. 5,171,574; U.S. Pat. No. 5,266,683; U.S. Pat.No. 5,354,557; and U.S. Pat. No. 5,468,845, the disclosures of which areherein incorporated by reference. Other of the aforementioned matriceshave also been well described in the art. See, for example, biomaterialstreatises such as LeGeros and Daculsi in Handbook of Bioactive Ceramics,II pp. 17–28 (1990, CRC Press); and other published descriptions such asYang Cao, Jie Weng Biomaterials 17, (1996) pp. 419–424; LeGeros, Adv.Dent. Res. 2, 164 (1988); Johnson et al., J. Orthopaedic Research, 1996,Vol. 14, pp. 351–369; and Piattelli et al., Biomaterials 1996, Vol. 17,pp. 1767–1770, the disclosures of which are herein incorporated byreference.

“Osteogenic device” is understood to mean a composition comprising atleast osteogenic protein dispersed in a matrix. As disclosed herein, an“improved osteogenic device” comprises osteogenic protein, a matrix asdefined above, and a binding agent as defined below. In contrast, a“standard osteogenic device” comprises osteogenic protein and a matrix,but not a binding agent; standard osteogenic devices can comprise eithera synthetic, polymeric or a matrix as defined above. In the Examples andteachings set forth below, standard osteogenic devices are furtherdesignated; standard devices, OP device, OP-1 device, or OP. Improvedosteogenic devices are further designated: CMC-containing device,CMC-containing standard device, CMC/OP-1 device, OP-1/CMC/collagen andOPCMC/collagen. As used herein, a “mock device” does not containosteogenic protein and is formulated free of any known osteoinductivefactor. The instant invention also contemplates improved devicescomprising at least two different osteogenic proteins and/or at leasttwo different matrices, as defined herein. Other embodiments of improveddevice can further comprise at least two different binding agents, asdefined herein. In still other embodiments, any one of theaforementioned improved devices can further comprise a wetting agent, asdefined herein. Any of the aforementioned embodiments can also includeradiopaque components, such as commercially available contrast agents.Generally, there are three well-known types of suchagents—hydroxyapatites, barium sulfate, and organic iodine. Devicescontaining radiopaque components are particularly useful for deviceadministration at a closed defect site, as discussed elsewhere herein.Identification of a suitable radiopaque component requires only ordinaryskill and routine experimentation. See, for example, radiographictreatises including, Ehrlich and McCloskey, Patient Care ini Radiography(Mosby Publisher, 1993); Carol, Fuch's Radiographic Exposure Processingand Quality Control (Charles C. Thomas Publisher, 1993); and Snopek,Fundamentals of Special Radiographic Procedures, (W. B. SaundersCompany, 1992), the disclosures of which are herein incorporated byreference.

Preferred embodiments of improved devices are adherent to bone,cartilage, muscle and/or other tissue. They have improved handlingproperties and are resistant to dislodging upon irrigation duringsurgery and upon suturing. Similarly, they are cohesive and not washedaway, disintegrated or diluted by irrigation and/or infiltrating bodyfluids such as blood. Preferred embodiments remain adherentpost-surgery, even at an articulating joint. Of particular importance isthat improved devices are readily confined to the defect site.Functionally, the improved osteogenic device of the instant inventioninduces accelerated bone and/or cartilage formation, as well as higherquality, more stable repair tissue and can achieve those benefits atdoses of osteogenic protein lower than required with a standardosteogenic device. Thus, the admixture of osteogenic protein withnon-synthetic, non-polymeric matrix and a binding agent has unexpectedproperties upon which the skilled practitioner can now capitalize asexemplified herein. One currently preferred embodiment comprises OP-1,collagen matrix and the binding agent carboxymethylcellulose (CMC). Asdiscussed below, an advantage associated with this currently preferredbinding agent, CMC, is its effectiveness even when present in lowrelative amounts. For example, in certain embodiments exemplifiedherein, OP-1 can be used in amounts ranging from approximately 1.25 to2.50 mg per approximately 1000 mg collagen and per approximately 180 to200 mg CMC.

In certain other embodiments, these amounts of protein, matrix andbinding agent can be increased or decreased according to the conditionsand circumstances related to defect repair. A wetting agent such assaline can be further added. As exemplified below, a preferredconfiguration for implantation at an open defect site assumes a puttyconsistency. It can be molded and shaped by the surgeon prior toimplantation. This configuration is achieved by adjusting the proportionof matrix to binding agent to wetting agent in a manner similar to thattaught herein. As further exemplified below, closed defects can betreated with a looser, more fluid device configuration resembling aviscous liquid. Such configurations can be injected without surgicalintervention at a defect site. Again, merely adjusting the proportionsof matrix to binding agent to wetting agent can achieve this embodiment.Currently, a preferred improved device comprises approximately 1 partbinding agent (w/w) to approximately 5 parts matrix (w/w). As describedherein below, other proportions can be used to prepare improved devices,depending upon the nature of binding agent and/or matrix.

Of course, an essential feature of any formulation of improvedosteogenic device is that it must be effective to provide at least alocal source of osteogenic protein at the defect site, even iftransient. As exemplified below, the binding agent content of animproved osteogenic device does not affect protein release/retentionkinetics. This is unexpected in view of contrary observations thatpolymer-containing standard devices failed to show clinicallysignificant osteoinducing effects in the absence of sequesteringmaterial (defined to include cellulosic materials) because proteindesorbtion was too great. (See, for example, U.S. Pat. No. 5,597,897.)As exemplified below, even when a binding agent as defined herein ispresent, protein is still desorbed from the improved device yetosteoinductive effects are readily apparent. While not wishing to bebound by theory, the unexpected features and benefits associated withthe instant invention appear to relate less to a protein-binding agentinteraction and more to a binding agent-matrix interaction.Specifically, binding agents as defined herein appear to complementand/or interact synergistically with the matrix required by the instantinvention. This has heretofore been unappreciated, and this combinationis discouraged by the teachings of the prior art. (See, for example,U.S. Pat. Nos. 5,520,923; 5,597,897; and WO 95/24210.)

The term “unitary” device refers to an improved osteogenic deviceprovided to the practitioner as a single, pre-mixed formulationcomprising osteogenic protein, matrix and binding agent. The term“non-unitary” device refers to an improved osteogenic device provided tothe practitioner in at least two separate packages for admixing prior touse.

Typically, a non-unitary device comprises at least binding agentpackaged separately from the osteogenic protein and the matrix. The term“carrier” refers to an admixture of binding agent and matrix, as each isdefined herein. Thus, for example, an improved osteogenic device asdisclosed herein comprises osteogenic protein and a carrier.

In addition to osteogenic proteins, various growth factors, hormones,enzymes, therapeutic compositions, antibiotics, or other bioactiveagents can also be contained within an improved osteogenic device. Thus,various known growth factors such as EGF, PDGF, IGF, FGF, TGF-α, andTGF-β can be combined with an improved osteogenic device and deliveredto the defect site. An improved osteogenic device can also be used todeliver chemotherapeutic agents, insulin, enzymes, enzyme inhibitorsand/or chemoattractant/chemotactic factors.

“Osteogenic protein”, or bone morphogenic protein, is generallyunderstood to mean a protein which can induce the full cascade ofmorphogenic events culminating in endochondral bone formation. Asdescribed elsewhere herein, the class of proteins is typified by humanosteogenic protein (hOP1). Other osteogenic proteins useful in thepractice of the invention include osteogenically active forms of OP1,OP2, OP3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, DPP, Vgl, Vgr, 60Aprotein, GDF-1, GDF-3, GDF-5, 6, 7, BMP10, BMP11, BMP13, BMP15, UNIVIN,NODAL, SCREW, ADMP or NEURAL and amino acid sequence variants thereof.In one currently preferred embodiment, osteogenic protein includes anyone of: OP1, OP2, OP3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acidsequence variants and homologs thereof, including species homologsthereof. Particularly preferred osteogenic proteins are those comprisingan amino acid sequence having at least 70% homology with the C-terminal102–106 amino acids, defining the conserved seven cysteine domain, ofhuman OP-1, BMP2, and related proteins. Certain preferred embodiments ofthe instant invention comprise the osteogenic protein, OP-1. Certainother preferred embodiments comprise mature OP-1 solubilized in aphysiological saline solution. As further described elsewhere herein,the osteogenic proteins suitable for use with Applicants' invention canbe identified by means of routine experimentation using theart-recognized bioassay described by Reddi and Sampath. “Amino acidsequence homology” is understood herein to mean amino acid sequencesimilarity. Homologous sequences share identical or similar amino acidresidues, where similar residues are conservative substitutions for, orallowed point mutations of, corresponding amino acid residues in analigned reference sequence. Thus, a candidate polypeptide sequence thatshares 70% amino acid homology with a reference sequence is one in whichany 70% of the aligned residues are either identical to, or areconservative substitutions of, the corresponding residues in a referencesequence. Examples of conservative variations include the substitutionof one hydrophobic residue, such as isoleucine, valine, leucine ormethionine, for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic foraspartic acids, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

Proteins useful in this invention include eukaryotic proteins identifiedas osteogenic proteins (see U.S. Pat. No. 5,011,691, incorporated hereinby reference), such as the OP-1, OP-2, OP-3 and CBMP-2 proteins, as wellas amino acid sequence-related proteins, such as DPP (from Drosophila),Vg1 (from Xenopus), Vgr-1 (from mouse), GDF-1 (from humans, see Lee(1991), PNAS 88:4250–4254), 60A (from Drosophila, see Wharton et al.(1991) PNAS 88:9214–9218), dorsalin-1 (from chick, see Basler et al.(1993) Cell 73:687–702 and GenBank accession number L12032) and GDF-5(from mouse, see Storm et al. (1994) Nature 368:639–643). BMP-3 is alsopreferred. Additional useful proteins include biosynthetic morphogenicconstructs disclosed in U.S. Pat. No. 5,011,691, e.g., COP-1, 3–5, 7 and16, as well as other proteins known in the art. Still other proteinsinclude osteogenically active forms of BMP-3b (see Takao, et al.,(1996), Biochem. Biophys. Res. Comm. 219: 656–662. BMP-9 (seeWO95/33830), BMP-15 (see WO96/35710), BMP-12 (see WO95/16035), CDMP-1(see WO 94/12814), CDMP-2 (see WO94/12814), BMP-10 (see WO94/26893),GDF-1 (see WO92/00382), GDF-10 (see WO95/10539), GDF-3 (see WO94/15965)and GDF-7 (WO95/01802).

Still other useful proteins include proteins encoded by DNAs competentto hybridize to a DNA encoding an osteogenic protein as describedherein, and related analogs, homologs, muteins (biosynthetic variants)and the like (see below). Certain embodiments of the improved osteogenicdevices contemplated herein comprise osteogenic protein functionallyand/or stably linked to matrix.

“Binding Agent”, as used herein, means any physiologically-compatiblematerial which, when admixed with osteogenic protein and matrix asdefined herein, promotes bone and/or cartilage formation. Preferredbinding agents promote such repair using less osteogenic protein thanstandard osteogenic devices. Among the other characteristics of apreferred binding agent is an ability to render the device: pliable,shapeable and/or malleable; injectable; adherent to bone, cartilage,muscle and other tissues; resistant to disintegration upon washingand/or irrigating during surgery; and, resistant to dislodging duringsurgery, suturing and post-operatively, to name but a few. Additionally,in certain preferred embodiments, a binding agent can achieve theaforementioned features and benefits when present in low proportions.For example, a currently preferred improved device comprisesapproximately 1 part binding agent and approximately 5 parts matrix.Another currently preferred device comprises approximately 3 partsbinding agent to 5 parts matrix. Certain binding agents can be used inequal or greater proportions relative to matrix, but, such agents shouldbe tested as taught below to identify possible matrix dilution effects.

Those binding agents contemplated as useful herein include, but are notlimited to: art-recognized suspending agents, viscosity-producing agentsand emulsifying agents. In particular, art-recognized agents, such ascellulose gum derivatives, sodium alginate, and gelatin powder can beused. More particularly, cellulosic agents such as alkylcelluloses, arepreferred including agents such as methylcellulose,methylhydroxyethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose, and hydroxyalkylcelluloses, to name but a few.Currently, the most preferred is carboxymethylcellulose, including thesodium salt thereof. As exemplified below, other binding agents suitablefor use in the instant invention include, but are not limited to,dextran, mannitol, white petrolatum, sesame oil and admixtures thereof.In view of the teachings set forth herein, the artisan can identifysuitable equivalents of the above-identified binding agents using merelyroutine experimentation and ordinary skill.

“Wetting Agent”, as used herein, means any physiologically-compatibleaqueous solution, provided it does not interfere with bone and/orcartilage formation. In certain embodiments of the instant invention,wetting agent is admixed with an improved device to achieve theconsistency necessitated by the mode of defect repair. As taught herein,wetting agent can be used to achieve a putty configuration or,alternatively, a viscous liquid configuration. A currently preferredwetting agent is physiological saline. Equivalents can be identified bythe artisan using no more than routine experimentation and ordinaryskill.

The means for making and using the methods, implants and devices of theinvention, as well as other material aspects concerning their nature andutility, including how to make and how to use the subject matterclaimed, will be further understood from the following, whichconstitutes the best mode currently contemplated for practicing theinvention. It will be appreciated that the invention is not limited tosuch exemplary work or to the specific details set forth in theseexamples.

I. PROTEIN CONSIDERATIONS

A. Biochemical, Structural and Functional Properties of Bone MorphogenicProteins

Naturally occurring proteins identified and/or appreciated herein to beosteogenic or bone morphogenic proteins form a distinct subgroup withinthe loose evolutionary grouping of sequence-related proteins known asthe TGF-β superfamily or supergene family. The naturally occurring bonemorphogens share substantial amino acid sequence homology in theirC-terminal regions (domains). Typically, the above-mentioned naturallyoccurring osteogenic proteins are translated as a precursor, having anN-terminal signal peptide sequence typically less than about 30residues, followed by a “pro” domain that is cleaved to yield the matureC-terminal domain. The signal peptide is cleaved rapidly upontranslation, at a cleavage site that can be predicted in a givensequence using the method of Von Heijne (1986) Nucleic Acids Research14:4683–4691. The pro domain typically is about three times larger thanthe fully processed mature C-terminal domain.

In preferred embodiments, the pair of morphogenic polypeptides haveamino acid sequences each comprising a sequence that shares a definedrelationship with an amino acid sequence of a reference morphogen.Herein, preferred osteogenic polypeptides share a defined relationshipwith a sequence present in osteogenically active human OP-1, SEQ ID NO:2. However, any one or more of the naturally occurring or biosyntheticsequences disclosed herein similarly could be used as a referencesequence. Preferred osteogenic polypeptides share a defined relationshipwith at least the C-terminal six cysteine domain of human OP-1, residues335–431 of SEQ ID NO: 2. Preferably, osteogenic polypeptides share adefined relationship with at least the C-terminal seven cysteine domainof human OP-1, residues 330–431 of SEQ ID NO: 2. That is, preferredpolypeptides in a dimeric protein with bone morphogenic activity eachcomprise a sequence that corresponds to a reference sequence or isfunctionally equivalent thereto.

Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differs from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy bone morphogenicactivity. Accordingly, conservative substitutions of corresponding aminoacids in the reference sequence are preferred. Amino acid residues thatare conservative substitutions for corresponding residues in a referencesequence are those that are physically or functionally similar to thecorresponding reference residues, e.g., that have similar size, shape,electric charge, chemical properties including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al. (1978), 5 Atlas of ProteinSequence and Structure, Suppl. 3, ch. 22 (pp. 354–352), Natl. Biomed.Res. Found., Washington, D.C. 20007, the teachings of which areincorporated by reference herein.

Examples of conservative substitutions include: Conservativesubstitutions typically include the substitution of one amino acid foranother with similar characteristics, e.g., substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

Natural-sourced osteogenic protein in its mature, native form is aglycosylated dimer typically having an apparent molecular weight ofabout 30–36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated peptide subunits having apparentmolecular weights of about 16 kDa and 18 kDa. In the reduced state, theprotein has no detectable osteogenic activity. The unglycosylatedprotein, which also has osteogenic activity, has an apparent molecularweight of about 27 kDa. When reduced, the 27 kDa protein gives rise totwo unglycosylated polypeptides, having molecular weights of about 14kDa to 16 kDa, capable of inducing endochondral bone formation in amammal. As described above, particularly useful sequences include thosecomprising the C-terminal 96 or 102 amino acid sequences of DPP (fromDrosophila), Vg1 (from Xenopus), Vgr-1 (from mouse), the OP1 and OP2proteins, proteins (see U.S. Pat. No. 5,011,691 and Oppermann et al., aswell as the proteins referred to as BMP2, BMP3, BMP4 (see WO88/00205,U.S. Pat. No. 5,013,649 and WO91/18098), BMP5 and BMP6 (see WO90/11366,PCT/US90/01630), BMP8 and BMP9.

Other morphogenic proteins useful in the practice of the inventioninclude morphogenically active forms of OP1, OP2, OP3, BMP2, BMP3, BMP4,BMP5, BMP6, BMP9, GDF-5, GDF-6, GDF-7, DPP, Vgl, Vgr, 60A protein,GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP10, BMP11, BMP13, BMP15, UNIVIN,NODAL, SCREW, ADMP or NURAL and amino acid sequence variants thereof. Inone currently preferred embodiment, osteogenic protein include any oneof: OP1, OP2, OP3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acid sequencevariants and homologs thereof, including species homologs, thereof.

Publications disclosing these sequences, as well as their chemical andphysical properties, include: OP-1 and OP-2: U.S. Pat. No. 5,011,691,U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085–2093;OP-3: WO94/10203 (PCT US93/10520); BMP2, BMP3, BMP4: WO88/00205, Wozneyet al. (1988) Science 242:1528–1534); BMP5 and BMP6: Celeste et al.(1991) PNAS 87: 9843–9847; Vgr-1: Lyons et al. (1989) PNAS 86:4554–4558; DPP: Padgett et al. (1987) Nature 325: 81–84; Vg-1: Weeks(1987) Cell 51: 861–867; BMP-9: WO95/33830 (PCT/US95/07084); BMP10:WO94/26893 (PCT/US94/05290); BMP-11: WO94/26892 (PCT/US94/05288); BMP12:WO95/16035 (PCT/US94/14030); BMP-13: WO95/16035 (PCT/US94/14030); GDF-1:WO92/00382 (PCT/US91/04096) and Lee et al. (1991) PNAS 88: 4250–4254;GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO94/15966 (PCT/US94/00685);GDF-10: WO95/10539 (PCT/US94/11440); GDF-11: WO96/01845(PCT/US95/08543); BMP-15: WO96/36710 (PCT/US96/06540); MP121: WO96/01316(PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO94/15949 (PCT/US94/00657) andWO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350); GDF-6(CDMP-2, BMP13): WO95/01801 (PCT/US94/07762) and WO96/14335 andWO95/10635 (PCT/US94/14030); GDF-7 (CDMP-3, BMP-12): WO95/10802(PCT/US94/07799) and WO95/10635 (PCT/US94/14030). In another embodiment,useful proteins include biologically active biosynthetic constructs,including novel biosynthetic morphogenic proteins and chimeric proteinsdesigned using sequences from two or more known morphogens. See also thebiosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, thedisclosure of which is incorporated herein by reference (e.g., COP-1,COP-3, COP-4, COP-5, COP-7, and COP-16).

In certain preferred embodiments, bone morphogenic proteins usefulherein include those in which the amino acid sequences comprise asequence sharing at least 70% amino acid sequence homology or“similarity”, and preferably 80% homology or similarity, with areference morphogenic protein selected from the foregoing naturallyoccurring proteins. Preferably, the reference protein is human OP-1, andthe reference sequence thereof is the C-terminal seven cysteine domainpresent in osteogenically active forms of human OP-1, residues 330–431of SEQ ID NO: 2. In certain embodiments, a polypeptide suspected ofbeing functionally equivalent to a reference morphogen polypeptide isaligned therewith using the method of Needleman, et al. (1970) J. Mol.Biol. 48:443–453, implemented conveniently by computer programs such asthe Align program (DNAstar, Inc.). As noted above, internal gaps andamino acid insertions in the candidate sequence are ignored for purposesof calculating the defined relationship, conventionally expressed as alevel of amino acid sequence homology or identity, between the candidateand reference sequences. “Amino acid sequence homology” is understoodherein to include both amino acid sequence identity and similarity.Homologous sequences share identical and/or similar amino acid residues,where similar residues are conservation substitutions for, or “allowedpoint mutations” of, corresponding amino acid residues in an alignedreference sequence. Thus, a candidate polypeptide sequence that shares70% amino acid homology with a reference sequence is one in which any70% of the aligned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence. Ina currently preferred embodiment, the reference sequence is OP-1. Bonemorphogenic proteins useful herein accordingly include allelic,phylogenetic counterpart and other variants of the preferred referencesequence, whether naturally-occurring or biosynthetically produced(e.g., including “muteins” or “mutant proteins”), as well as novelmembers of the general morphogenic family of proteins, including thoseset forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

In other preferred embodiments, the family of bone morphogenicpolypeptides useful in the present invention, and members thereof, aredefined by a generic amino acid sequence. For example, Generic Sequence7 (SEQ ID NO: 4) and Generic Sequence 8 (SEQ ID NO: 5) disclosed below,accommodate the homologies shared among preferred protein family membersidentified to date, including at least OP-1, OP-2, OP-3, CBMP-2A,CBMP-2B, BMP-3, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1. The aminoacid sequences for these proteins are described herein and/or in theart, as summarized above. The generic sequences include both the aminoacid identity shared by these sequences in the C-terminal domain,defined by the six and seven cysteine skeletons (Generic Sequences 7 and8, respectively), as well as alternative residues for the variablepositions within the sequence. The generic sequences provide anappropriate cysteine skeleton where inter- or intramolecular disulfidebonds can form, and contain certain critical amino acids likely toinfluence the tertiary structure of the folded proteins. In addition,the generic sequences allow for an additional cysteine at position 36(Generic Sequence 7) or position 41 (Generic Sequence 8), therebyencompassing the morphogenically active sequences of OP-2 and OP-3.

Generic Sequence 7             Leu Xaa Xaa Xaa Phe Xaa Xaa              1               5 Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro         10                   15 Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly         20                   25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa         30                   35 Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa         40                   45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa         50                   55 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa         60                   65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa         70                   75 Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa         80                   85 Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa         90                   95wherein each Xaa independently is selected from a group of one or morespecified amino acids defined as follows: “Res.” means “residue” and Xaaat res.2=(Tyr or Lys); Xaa at res;3=Val or Ile); Xaa at res.4=(Ser, Aspor Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp orGlu); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His,Asn or Ser); Xaa at res. 12=(Asp, Arg, Asn or Glu); Xaa at res. 13=(Trpor Ser); Xaa at res.15=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa atres.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaaat res. 19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala,Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr or Phe); Xaa atres.26=(Glu, His, Tyr, Asp, Gln, Ala or Ser); Xaa at res.28=(Glu, Lys,Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa atres.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa atres.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu,Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa atres.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa atres.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa atres.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa atres.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.48=(Leuor Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asn or Arg);Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met,Asn, Ala, Val, Gly or Leu); Xaa at res.53=(Asn, Lys, Ala, Glu, Gly orPhe); Xaa at res.54=(Pro, Ser or Val); Xaa at res.55=(Glu, Asp, Asn,Gly, Val, Pro or Lys); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser,Gly, Ile or His); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro orAsp); Xaa at res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala);Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala) and Xaa at res.97=(His or Arg).

Generic Sequence 8 (SEQ ID NO: 5) includes all of Generic Sequence 7 andin addition includes the following sequence (SEQ ID NO: 8) at itsN-terminus:

Cys Xaa Xaa Xaa Xaa   1                5Accordingly, beginning with residue 7, each “Xaa” in Generic Sequence 8is a specified amino acid defined as for Generic Sequence 7, with thedistinction that each residue number described for Generic Sequence 7 isshifted by five in Generic Sequence 8. Thus, “Xaa at res.2=(Tyr or Lys)”in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence 8. InGeneric Sequence 8, Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa atres.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); and Xaa atres.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

In another embodiment, useful osteogenic proteins include those definedby Generic Sequences 9 and 10, defined as follows.

Specifically, Generic Sequences 9 and 10 are composite amino acidsequences of the following proteins: human OP-1, human OP-2, human OP-3,human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, humanBMP-8, human BMP-9, human BMP10, human BMP-11, Drosophila 60A, XenopusVg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouseGDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouseGDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp, DrosophilaSCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10,human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b. Like GenericSequence 7, Generic Sequence 9 accommodates the C-terminal six cysteineskeleton and, like Generic Sequence 8, Generic Sequence 10 accommodatesthe seven cysteine skeleton.

Generic Sequence 9 (SEQ ID NO: 6) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1               5                    10 Xaa Xaa Xaa Xaa Xaa Xaa ProXaa Xaa Xaa                 15                   20 Xaa Xaa Xaa Xaa CysXaa Gly Xaa Cys Xaa                 25                   30 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa                 35                   40 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 45                  50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                55                   60 Xaa Cys Xaa Pro Xaa Xaa Xaa XaaXaa Xaa                 65                   70 Xaa Xaa Leu Xaa Xaa XaaXaa Xaa Xaa Xaa                 75                   80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa                 85                   90 Xaa XaaXaa Cys Xaa Cys Xaa                 95wherein each Xaa is independently selected from a group of one or morespecified amino acids define as follows: “Res.” means “residue” and Xaaat res. 1=(Phe, Leu or Glu); Xaa at res.2=(Tyr, Phe, His, Arg, Thr, Lys,Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa atrest.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa atres.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu,Leu, Ala or Gln): Xaa at res.8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gln); Xaa at res 52=(Ile, Met, Leu, Val, Lys,Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala, Gln,Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaa atres. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His); Xaaat res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaa atres. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly, Ser,Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg or Gly);Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys, Val orSer); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala, Glu,Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val); Xaaat res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly, Thr,Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaa atres. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr, Asnor Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr, Phe,Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile, Lys, Glnor Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res. 77=(Asp,Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser, Asn,Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res. 79=(Ser,Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn, Lys,Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thr orAla); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa at res.83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe, Thr,Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val); Xaaat res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln, Pro orLys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa at res.89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys); Xaa atres 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu, Val, Ala,Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr); Xaa atres. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu or Ser).Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile; after res.54 in GDF-1 there is a T; after res. 54 in BMP3 there is a V; after res.78 in BMP-8 and Dorsalin there is a G; after res. 37 in hGDF-1 there isPro, Gly, Gly, Pro.

Generic Sequence 10 (SEQ ID NO: 7) includes all of Generic Sequence 9(SEQ ID NO: 6) and in addition includes the following sequence (SEQ IDNO: 9) at its N-terminus:

Cys Xaa Xaa Xaa Xaa SEQ ID NO:9   1               5Accordingly, beginning with residue 6, each “Xaa” in Generic Sequence 10is a specified amino acid defined as for Generic Sequence 9, with thedistinction that each residue number described for Generic Sequence 9 isshifted by five in Generic Sequence 10. Thus, “Xaa at res. 1=(Tyr, Phe,His, Arg, Thr, Lys, Gln, Val or Glu)” in Generic Sequence 9 refers toXaa at res. 6 in Generic Sequence 10. In Generic Sequence 10, Xaa atres. 2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res. 3=(Lys,Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln, Arg,Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr, His, Arg,Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

As noted above, certain currently preferred bone morphogenic polypeptidesequences useful in this invention have greater than 60% identity,preferably greater than 65% identity, with the amino acid sequencedefining the preferred reference sequence of hOP-1. These particularlypreferred sequences include allelic and phylogenetic counterpartvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein. Accordingly, in certain particularly preferred embodiments,useful morphogenic proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (SEQ ID NO: 3), which defines the seven cysteineskeleton and accommodates the homologies between several identifiedvariants of OP-1 and OP-2. As described therein, each Xaa at a givenposition independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2.

Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa Asp Trp1                5                    10                    15 Xaa IleAla Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro    20                   25                   30                   35Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val HisXaa         40                   45                   50                  55 Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala ProThr Xaa Leu Xaa Ala             60                  65                  70 Xaa Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val IleLeu Xaa Lys Xaa Arg 75                   80                   85                  90 Asn Met Val Val Xaa Ala Cys Gly Cys His         95                  100wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaa atres. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ileor Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaaat res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser orThr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa atres. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

In still another preferred embodiment, useful osteogenically activeproteins have polypeptide chains with amino acid sequences comprising asequence encoded by a nucleic acid that hybridizes, under low, medium orhigh stringency hybridization conditions, to DNA or RNA encodingreference morphogen sequences, e.g., C-terminal sequences defining theconserved seven cysteine domains of OP-1, OP-2, BMP2, 4, 5, 6, 60A,GDF3, GDF6, GDF7 and the like. As used herein, high stringenthybridization conditions are defined as hybridization according to knowntechniques in 40% formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDSat 37° C. overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C.Standard stringence conditions are well characterized in commerciallyavailable, standard molecular cloning texts. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984): Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); and B. Perbal, A Practical Guide To MolecularCloning (1984).

As noted above, proteins useful in the present invention generally aredimeric proteins comprising a folded pair of the above polypeptides.Such morphogenic proteins are inactive when reduced, but are active asoxidized homodimers and when oxidized in combination with others of thisinvention to produce heterodimers. Thus, members of a folded pair ofmorphogenic polypeptides in a morphogenically active protein can beselected independently from any of the specific polypeptides mentionedabove.

The bone morphogenic proteins useful in the materials and methods ofthis invention include proteins comprising any of the polypeptide chainsdescribed above, whether isolated from naturally-occurring sources, orproduced by recombinant DNA or other synthetic techniques, and includesallelic and phylogenetic counterpart variants of these proteins, as wellas muteins thereof, and various truncated and fusion constructs.Deletion or addition mutants also are envisioned to be active, includingthose which may alter the conserved C-terminal six or seven cysteinedomain, provided that the alteration does not functionally disrupt therelationship of these cysteines in the folded structure. Accordingly,such active forms are considered the equivalent of the specificallydescribed constructs disclosed herein. The proteins may include formshaving varying glycosylation patterns, varying N-termini, a family ofrelated proteins having regions of amino acid sequence homology, andactive truncated or mutated forms of native or biosynthetic proteins,produced by expression of recombinant DNA in host cells.

The bone morphogenic proteins contemplated herein can be expressed fromintact or truncated cDNA or from synthetic DNAs in prokaryotic oreukaryotic host cells, and purified, cleaved, refolded, and dimerized toform morphogenically active compositions. Currently preferred host cellsinclude, without limitation, prokaryotes including E. coli, oreukaryotes including yeast, or mammalian cells, such as CHO, COS or BSCcells. One of ordinary skill in the art will appreciate that other hostcells can be used to advantage. Detailed descriptions of the bonemorphogenic proteins useful in the practice of this invention, includinghow to make, use and test them for osteogenic activity, are disclosed innumerous publications, including U.S. Pat. Nos. 5,266,683 and 5,011,691,the disclosures of which are incorporated by reference herein, as wellas in any of the publications recited herein, the disclosures of whichare incorporated herein by reference.

Thus, in view of this disclosure and the knowledge available in the art,skilled genetic engineers can isolate genes from cDNA or genomiclibraries of various different biological species, which encodeappropriate amino acid sequences, or construct DNAs fromoligonucleotides, and then can express them in various types of hostcells, including both prokaryotes and eukaryotes, to produce largequantities of active proteins capable of stimulating endochondral bonemorphogenesis in a mammal.

II. BINDING AGENT CONSIDERATIONS

As already explained, “binding agent”, as used herein, means anyphysiologically-compatible material which, when admixed with osteogenicprotein and matrix as defined herein promotes bone and/or cartilageformation. In certain currently preferred embodiments, binding agentspromote such repair using less osteogenic protein than standardosteogenic devices. Among the other characteristics of a preferredbinding agent is an ability to render the device: pliable, shapeableand/or malleable; injectable; adherent to bone, cartilage, muscle andother tissues; resistant to disintegration upon washing and/orirrigating during surgery; and, resistant to dislodging during surgery,suturing and post-operatively, to name but a few. Additionally, in acurrently preferred embodiment, binding agent can achieve theaforementioned features and benefits when present in relatively lowproportions. For example, a currently preferred improved devicecomprises approximately 1 part binding agent and approximately 5 partsmatrix. Another currently preferred device comprises 1 part bindingagent and 3 parts matrix. As exemplified herein, improved devices ofwidely divergent proportions can induce bone and cartilage such agents.For example, binding agent candidates are those described as useful asemulsifying agents, gel-forming agents, binders, or viscosity-producingagents for injectables and parenterals. Other candidates are agents usedto suspend ingredients for topical, oral or parenteral administration.Yet other candidates are agents useful as tablet binders, disintegrantsor emulsion stabilizers. Still other candidates are agents used incosmetics, toiletries and food products. When used for any of theforegoing applications, candidate agents are described as typicallypresent for conventional applications at concentrations ranging fromapproximately 0.1 to 6.0%. At the highest standard concentrations(4–6%), certain of the foregoing candidate agents are used in thepharmaceutical industry, for example, to produce medicaments in the formof gels or pastes.

Thus the skilled artisan can identify binding agent candidatesaccordingly and can similarly recognize equivalents of the preferredbinding agents specifically identified herein using only routine skilland routine experimentation. Having identified a suitable candidate(s),the skilled artisan can then follow the guidelines set forth below as tofinal selection of a preferred binding agent.

Based on studies similar to those described herein, examples of suitablebinding agents useful in the improved devices disclosed herein include,but are not limited to: mannitol/dextran combination; dextran alone;mannitol/white petrolatum combination; and sesame oil. Amannitol/dextran-containing improved device was formulated as follows.One part dextran 40, 3 parts mannitol, 1 part OP device. Such improveddevices were formulated with 2.5 mg osteogenic protein per g collagen orper 0.5 g collagen, thereby varying the dose of osteogenic protein. Foruse in the instant method, the formulation was wetted with approximately0.8 ml saline per 2.5 g mannitol/dextran-containing device. Next, adextran alone-containing device was formulated from either 4 partsdextran or 1 part dextran to 1 part OP device, and wetted withapproximately 0.8 ml saline per 2.0 g device. Dextran can range from3,000 to 40,000 m.w. Next, a mannitol/white petrolatum device wasformulated from 1.5 parts mannitol, 1.5 parts petrolatum, and 1 part OPdevice. This formulation does not require wetting. Finally, a sesameoil-containing improved device was formulated from 1 part oil and 1 partOP device. This formulation does not require wetting. Theabove-described improved devices illustrate the range of: specificbinding agents, proportions in improved devices, and volumes of wettingagent which can be used in the improved devices of the instantinvention. Chemistries, proportions and wetting requirements are varied,yet all are within the skill of the art. Each of the aforementionedimproved devices induced bone formation (as measured by calcium contentand % bone) when tested in the rat subcutaneous bioassay describedherein.

As taught herein, carboxymethylcellulose (CMC) is a currently preferredbinding agent. CMC is commercially available from suppliers such as, butnot limited to: Hercules Inc., Aqualon®Division, Delaware; FMCCorporation, Pennsylvania; British Celanese, Ltd., United Kingdom; andHenkel KGaA, United Kingdom. Carboxymethylcellulose sodium is the sodiumsalt of a polycarboxymethyl ether of cellulose with a typical molecularweight ranging from 90,000–700,000. CMC was identified as a candidatebinding agent based, in part, on the following: CMC is widely used inoral and topical pharmaceutical formulations as a viscosity-increasingagent. CMC is also used in cosmetics, toiletries and foods as anemulsifying agent (0.25–1.0%), gel forming agent (4.0–6.0%), injectable(0.05–0.75%), and tablet binder (1.0–6.0%).

While the foregoing characteristics are suggestive of suitability as abinding agent, the experiments detailed below confirmed that CMC wassuitable for use in the improved osteogenic devices disclosed herein.Such confirmatory experiments were necessary because none of theaforementioned applications are similar to the repair of bone orcartilage for which the improved osteogenic devices disclosed herein areuseful. For example, none of the aforementioned applications require CMCabove 6%, yet a currently preferred implantable improved device of theinstant invention comprises more than approximately 6% (w/w) CMC andpreferably at least approximately 10%, more preferably approximately12–20%, with approximately about 16% (w/w) or 1 part CMC to 5 partsstandard osteogenic device being most currently preferred for animplantable device. These approximate percentages are based oncalculations of total weight of matrix admixed with binding agent,excluding osteogenic protein and wetting agent.

Of significance to practice of the instant invention is the fact thatvarious grades of carboxymethylcellulose sodium are commerciallyavailable which have differing viscosities. Viscosities of variousgrades of carboxymethylcellulose sodium are reported and shown in Table1 below (see, Handbook of Pharmaceutical Excipients (2nd Edition),American Pharmaceutical Association & Royal Pharmaceutical Society ofGreat Britain).

TABLE 1 Standard Viscosity Grades of CarboxymethylcelluloseConcentration Viscosity Grade (% w/v) (cP) Low viscosity 4  50–200Medium viscosity 2 400–800 High viscosity 1 1500–3000

A number of grades of carboxymethylcellulose are commercially available,the most frequently used grade having a degree of substitution (DS) of0.7. The DS is defined as the average number of hydroxyl groupssubstituted per anhydroglucose unit. It is this DS which determines theaqueous solubility of the polymer. The degree of substitution and thestandard viscosity of an aqueous solution of stated concentration isindicated on any carboxymethylcellulose sodium labelling. Low viscosityCMC (Aqualon® Divison, Hercules Inc., Wilmington, Del.) is currentlypreferred. The currently preferred degrees of substitution range from0.65–0.90 (DS=0.7, Aqualon® Type 7L).

As described above, CMC is available in several grades—low, medium andhigh viscosity. In this regard, the viscosity of thecarboxymethylcellulose (CMC) used to formulate an improved osteogenicdevice was determined to be critical for bone formation. Contrary toteachings in the art, it has now been discovered that high viscosity CMCadversely affects bone formation when used in an improved osteogenicdevice comprising a matrix as defined herein. U.S. Pat. No. 5,587,897(“the '897 Patent”) teaches the use of high viscosity (2480 cP) (seeTable 1 above) CMC to induce bone formation. The devices in the '897Patent, however, require a synthetic polymer matrix, rather than abiological matrix such as collagen. Unexpectedly, when a biologicalmaterial such as collagen is used as a matrix, the improved device mustbe formulated with low viscosity CMC (approximately 10–50 cP, or 50–200)in order to induce bone and/or cartilage formation, as taught herein.

Toxicity Study Using a CMC Device

A toxicity study was conducted comparing a CMC-containing improveddevice to that of a standard device. The standard device was preparedwith 2.5 mg OP-1/gram collagen matrix. The CMC containing improveddevice was prepared by adding low viscosity CMC (Aqualon®) to a standarddevice at the ratio of 1:5 followed by irradiation. 25 mg aliquots of astandard device or mock device (i.e., no osteogenic protein) and 30 mgaliquots of CMC containing improved device or mock CMC device wereimplanted at a rat sub-cutaneous site as described elsewhere herein (oneimplant per animal). Three implants from each formulation were removedat 7 days, 14 days, 21 days and 28 days post-implantation, and evaluatedhistologically for bone and cartilage formation and for local tissuereaction. No adverse cellular reaction was observed, and there was noevidence to indicate any adverse effects of CMC, as deterimed byevaluating inflammation and fibrous formation. The histological profileof the CMC-containing-improved device was generally similar to thestandard OP-device. Serum calcium and alkaline phosphatase levels,measured using standard teachings also followed that of the standardosteogenic device. Finally, using standard toxicity analyses on immatureand mature rats, no significant lesions were detected.

Improved Device Bioactivity Studies

Based on a series of routine studies described below, the bioactivity ofa standard osteogenic device was not adversely affected by admixturewith CMC. Rather, bioactivity is at least comparable for both deviceconfigurations, but the ability to manipulate the deviceintraoperatively and to retain the device at the defect site duringsurgery and wound closure is enhanced by CMC. For these studies,irradiated CMC was added to the standard device prior to implantation.

Briefly, two studies were conducted measuring the in vivo release ofOP-1 from a standard device +/−CMC. In one experiment, 75 mg ofirradiated device +/−15 mg of irradiated CMC were implanted in asubcutaneous site in rats as described herein. The implanted deviceswere removed 1 hour, 1 day, 3 days and 6 days after implantation,followed by extraction with 8M urea buffer; the OP-1 content wasanalyzed by routine ELISA and western blot analysis. OP-1 device (notimplanted into the animals) was extracted with 8M urea buffer and usedas the internal standard. In general, the kinetics of in vivo release ofOP-1 from the standard OP device and the CMC-containing improved devicewere similar. The observation that there is no difference in OP-1sequestration or retention by a standard device versus an improveddevice containing a combination of collagen matrix and CMC is anunexpected result. It has been reported that, when combined withnon-biological polymeric matrices, CMC acts to sequester osteogenicprotein. (See, for example, U.S. Pat. No. 5,597,897).

In vitro studies were also conducted. In these studies, released OP-1was measured in contrast to the above-described in vivo studies, inwhich OP-1 remaining in the device was measured. In one study, 25 mg ofOP device or CMC device was wetted with saline. 1 ml of bovine serum wasthen added to each device, and the devices were incubated at 37° C. Thesupernatant was removed and replaced with fresh serum at 1 and 3 hours.At 6 hours 8M urea was added to extract any OP-1 still associated withthe device. OP-1 concentrations in the supernatants were analyzed byroutine ELISA and western blot techniques. Both the standard OP deviceand the CMC-containing improved device had similar protein releasekinetics for the six hours studied. Again, these results were unexpectedin view of earlier reports that CMC acts to sequester osteogenic proteinand thereby retard and/or prevent its release from admixtures withsynthetic, polymeric matrices. (See, for example, U.S. Pat. No.5,597,897).

In conclusion, CMC does not substantially inhibit the retention orrelease of OP-1 from a collagen matrix—containing osteogenic device invivo or in vitro.

Stability Studies

A study (see Table 2) was conducted comparing the stability of thestandard OP device to a standard device containing CMC. Based on both invitro analyses and a bone-forming bioassay (described elsewhere herein),the CMC-containing improved device was observed to be at least as stableas the standard device when stored at 30 degrees for one year. The dataalso suggest that CMC can be premixed with the standard OP device andterminally sterilized for a unitary product configuration. Such aunitary product is useful for repair of local bone and cartilage defectsas exemplified below.

TABLE 2 Stability Of Various Osteogenic Device Formulations OP-1 OP-1OP-1 OP-1 Pre-Irradiation Post-Irradiation Recovery Recovery RecoveryRecovery OP-1 OP-1 after 4 after 3 after 6 after 12 FormulationRecovery* Recovery weeks months months months Standard 91% 63% 66% 58%47.4% 37.4% Device CMC Device 77% 57% 53% 50% 43.5% 34.8% *Based ontheoretical OP-1 content of 2.5 mg/gram

During formulation of a standard device containing CMC, the CMC andosteogenic proteins may be sterilized separately, for example, byexposure to gamma irradiation and then the sterilized componentscombined to produce the standard device containing CMC. Furthermore, theCMC can be premixed with the standard OP device and the resultingformulation sterilized, for example, by exposure to gamma irradiation.The latter process is referred to in the art as terminal sterilizationand has been used to sterilize other osteogenic devices. See, forexample, PCT/US96/10377, published Dec. 19, 1996, and U.S. Ser. No.08/478,452 now pending, the disclosures of which is incorporated byreference. As used herein, the terms “sterilization” and “sterilized”refer to a process using either physical or chemical means foreliminating substantially all viable organisms, especiallymicro-organisms, viruses and other pathogens, associated with the deviceof the invention. The sterilized devices of the invention preferablyhave a sterility assurance level of 10−⁶ as determined by Federal DrugAdministration (FDA) standards. In the case of gamma irradiated devices,for example, the appropriate dosages of irradiation necessary forsterilizing a particular device can be determined readily by consultingthe reference text “Associate for the Advancement of MedicalInstrumentation Guidelines,” published 1992. Guidelines are providedtherein for determining the radiation dose necessary to achieve a givensterility assurance level for a particular bioburden of the device.Dosages for sterilizing devices of the invention preferably are withinthe range of about 0.5 to about 4.0 megarods and most preferably arewithin the range of about 2.0 to about 3.5 megarods.

Additionally, a study was conducted to evaluate the short term stabilityof an osteogenic device to which CMC and saline had been added. Thestudy used a standard device to which 200 mg of separately packaged,irradiated CMC was added. Samples of CMC-containing improved device wereremoved and wet with saline. At 0, 1, 3, 6 and 22 hours the OP-1 wasextracted with 8 M urea buffer and analyzed by reverse-phase HPLC underreducing conditions. The extracts were also analyzed for OP-1 biologicalactivity a standard cell-based assay for measuring alkaline phosphatase.The data indicated that OP-1 retains biological activity under theseconditions. These data also suggested that the configuration ofCMC-containing improved device resulting from admixture of thesecomponent parts (standard osteogenic device/CMC/saline) is useable forseveral hours after it has been prepared, providing the practitionerwith significant intraoperative time during which the product remainsefficacious.

Testing of Binding Agent Integrity and Other Characteristics

Art-recognized USP methods were used for identification andcharacterization of bulk binding agents such as CMC. Tests includedtests for chemical identity, viscosity, pH, loss on drying and heavymetals. Material was also tested for bioburden prior to sterilization,as well as endotoxins, pH, appearance and sterility after irradiation. Astability study was conducted to monitor the viscosity, appearance andpH of the irradiated material. All levels and characteristics wereacceptable as determined using standard methods and techniques.

For example, CMC (Aqualon®-low viscosity) was evaluated for bioburdenand endotoxin content. Aqualon® CMC, Lot FP10 12342, was evaluated forthe presence of endotoxins (LAL) using the Kinetic Chromogenic LAL assayfrom BioWhittaker (Walkersville, Md., 21793).

“Bioburden” can be measured as follows. For example 200 mg samples ofCMC were solubilized in 100 ml of phosphate buffered water and filteredthrough 0.45 μm filters. The filters were placed on a TSA plate andincubated for 48 hours. Two samples of solubilized CMC were inoculatedwith 10–100 CFUs of Bacillus subtilis to be used as growth controls. Thedata suggest that the bioburden of the CMC is low, and that CMC does notinterfere in the analysis by killing bacteria or inhibiting cell growth.

CMC Characterization Post-Irradiation

A study was conducted comparing the viscosity of CMC pre- andpost-irradiation (gamma irradiation, 2.5–3.0 mega rads). The dataindicated that, as reported in the art, viscosity decreases afterirradiation. While this does not affect bioactivity or its overallutility as a binding agent (see studies set forth herein), the skilledpractitioner should take this feature into consideration when assessingviscosity or fluidity properties of an improved osteogenic device. Astudy was also conducted to evaluate the stability of irradiated CMC.The results indicated that irradiated CMC was stable for at least sixmonths at both 4 and 30° C. Viscosity was measured as the parameter ofstability. Similar analyses and assessments can be carried out for otherbinding agents or device materials used in a desired formulation.

III. FORMULATION AND DELIVERY CONSIDERATIONS

General Considerations

The devices of the invention can be formulated using routine methods.All that is required is determination of the desired final concentrationof osteogenic protein per device, keeping in mind that the deliveredvolume of device can be, but is not necessarily required to be, lessthan the volume at the defect site. The desired final concentration ofprotein will depend on the specific activity of the protein as well asthe type, volume, and/or anatomical location of the defect.Additionally, the desired final concentration of protein can depend onthe age, sex and/or overall health of the recipient. Typically, for acritical size segmental defect approximately at least 2.5 cm in length,0.5–1.75 mg osteogenic protein has been observed using the standarddevice to induce bone formation sufficient to repair the gap. In thecase of a non-critical size defect or a fresh fracture, approximately0.1–0.5 mg protein has been observed using the standard osteogenicdevice to repair the defect. Optimization of dosages requires no morethan routine experimentation and is within the skill level of one ofordinary skill in the art.

As exemplified herein, osteogenic protein and a binding agent such ascarboxymethylcellulose (low viscosity, Aqualon®) can be admixed to forma putty. In some embodiments, saline is added to binding agent to form apaste or putty in which an osteogenic protein such as OP-1 is dispersed.A paste configuration can be used to paint the surfaces of a defect,such as a cavity. Pastes can be used to paint fracture defects, chondralor osteochondral defects, as well as bone defects at a prostheticimplant site. A more fluid configuration can be injected or extrudedinto or along the surfaces of a defect, in a manner similar to extrudingtoothpaste or caulking from a tube, such that a bead of device isdelivered along the length of the defect site. Typically, the diameterof the extruded bead is determined by the type of defect as well as thevolume of the void at the defect site.

As mentioned above, other binding agents as defined herein can be usedto formulate a device with a configuration like putty. As will beobvious to the skilled artisan, such a configuration results fromadjusting the proportion of carrier to wetting agent, with less wettingagent producing a drier device and more producing a wetter device. Theprecise device configuration suitable to repair a defect will at leastdepend on the type of defect and the size of the defect. The skilledartisan will appreciate the variables.

Binding Agent Formulation Studies

Based on the following type of studies, it was established thatapproximately 0.2 g of CMC to approximately 1.0 g standard osteogenicdevice yields an improved device with the currently preferred handlingproperties. Varying ratios of CMC and collagen were combined and thenwet with saline. Each resulting mixture of CMC and matrix was suspendedin a 15 ml conical centrifuge tube of water and placed on a rotaryshaker (100 rpm). Settling time was recorded when loosened or releasedcollagen matrix particles settled to a predetermined mark on the tube.The data summarized in Table 3 and FIG. 1 suggest that a range ofapproximately 0.15 to 0.25 g CMC/g collagen can maximize cohesiveness,integrity and handling properties.

TABLE 3 Effect Of CMC/Collagen Ratio On Dispersion Time g CMC/g CollagenSettling Time 0.20 g 19 mm 0.19 17 0.18 6 0.15 4 0.12 0.5 sec

The preferred amount of saline for wetting the CMC device was alsostudied. In this study, approximately 0.2 g of CMC were mixed withapproximately 1 g standard osteogenic device. Varying amounts of salinewere added, and the consistency of the resulting device was noted. Thequalitative and quantitative results from this study are summarized inTable 4 and FIG. 2, respectively. Generally, these data illustrate thatthere is a range of wetting agent volumes which can accommodate thepractitioner while enabling the device to retain its integrity andcohesiveness. For a binding agent like CMC, the data suggest that morethan approximately 1.5 ml, approximately 1.8 to 2.5 ml of saline, is thecurrently preferred wetting volume (for approximately 1 gram of deviceadmixed with approximately 200 mg of a binding agent such as CMC) toachieve an implantable device with the currently preferred puttyconsistency. Amounts of saline in excess of this achieve an injectabledevice with the currently preferred fluid consistency. As exemplifiedelsewhere herein, an implantable device configuration is suitable foruse at an open defect site, while an injectable device configuration issuitable for use at a closed defect site. In terms of gram equivalents,approximately 0.5 g to approximately 3.0 g saline has been determined toyield improved devices with desirable consistencies; the higher theweight, the more injectable is the configuration.

TABLE 4 Wetting of the CMC-Containing Device Amount of 1 gram StandardDevice Saline plus 200 mg CMC Added Observations 1.5 ml Dry 1.75 Rollsinto a ball; paste 2.0 Currently preferred handling consistency; putty.Rolls into a ball. 2.5 Acceptable handling consistency; stillputty-like. 2.75 Leaves small particles of matrix on vessel wall. 3.0Sticky; soft paste. 3.5 Sticky; soft paste. 3.75 Loose paste. 4.0Consistency same as above. 4.25 Liquid

In certain embodiments of the present invention, preparation of theactual improved osteogenic device can occur immediately prior to itsdelivery to the defect site. As exemplified herein, CMC-containingimproved devices can be prepared on-site, suitable for admixingimmediately prior to surgery. In one embodiment, low viscosity CMC(Aqualon®) was packaged and irradiated separately from the osteogenicprotein OP-1 and collagen matrix. The OP-1 protein in collagen matrixthen was admixed with the binding agent. Devices prepared in this mannerwere observed to be at least as biologically active as the standarddevice without CMC.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

IV. BIOASSAY

A. Bioassay of Osteogenic Activity: Endochondral Bone Formation andRelated Properties

The following sets forth exemplary protocols for identifying andcharacterizing bona fide osteogenic or bone morphogenic proteins as wellas osteogenic devices within the scope of Applicants' invention.

The art-recognized bioassay for bone induction as described by Sampathand Reddi (Proc. Natl. Acad. Sci. USA (1983) 80:6591–6595) and U.S. Pat.No. 4,968,590, the disclosures of which are herein incorporated byreference, is used to establish the efficacy of the purificationprotocols. Briefly, this assay consists of depositing the test samplesin subcutaneous sites in allogenic recipient rats under etheranesthesia. A vertical incision (1 cm) is made under sterile conditionsin the skin over the thoracic region, and a pocket is prepared by bluntdissection. In certain circumstances, approximately 25 mg of the testsample is implanted deep into the pocket and the incision is closed witha metallic skin clip. The heterotropic site allows for the study of boneinduction without the possible ambiguities resulting from the use oforthotopic sites.

The sequential cellular reactions occurring at the heterotropic site arecomplex. The multistep cascade of endochondral bone formation includes:binding of fibrin and fibronectin to implanted matrix, chemotaxis ofcells, proliferation of fibroblasts, differentiation into chondroblasts,cartilage formation, vascular invasion, bone formation, remodeling, andbone marrow differentiation.

In rats, this bioassay model exhibits a controlled progression throughthe stages of matrix induced endochondral bone development including:(1) transient infiltration by polymorphonuclear leukocytes on day one;(2) mesenchymal cell migration and proliferation on days two and three;(3) chondrocyte appearance on days five and six; (4) cartilage matrixformation on day seven; (5) cartilage calcification on day eight; (6)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (7) appearance of osteoblastic and bone remodelingon days twelve to eighteen; and (8) hematopoietic bone marrowdifferentiation in the ossicle on day twenty-one.

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Staining with toluidine blue orhemotoxylin/eosin clearly demonstrates the ultimate development ofendochondral bone. Twelve day bioassays are sufficient to determinewhether bone inducing activity is associated with the test sample.

Additionally, alkaline phosphatase activity can be used as a marker forosteogenesis. The enzyme activity can be determinedspectrophotometrically after homogenization of the excised testmaterial. The activity peaks at 9–10 days in vivo and thereafter slowlydeclines. Samples showing no bone development by histology should haveno alkaline phosphatase activity under these assay conditions. The assayis useful for quantitation and obtaining an estimate of bone formationvery quickly after the test samples are removed from the rat. Forexample, samples containing osteogenic protein at several levels ofpurity have been tested to determine the most effective dose/puritylevel, in order to seek a formulation which could be produced on anindustrial scale. The results as measured by alkaline phosphataseactivity level and histological evaluation can be represented as “boneforming units”. One bone forming unit represents the amount of proteinthat is needed for half maximal bone forming activity on day 12.Additionally, dose curves can be constructed for bone inducing activityin vivo at each step of a purification scheme by assaying variousconcentrations of protein. Accordingly, the skilled artisan canconstruct representative dose curves using only routine experimentation.

B. Collagen Formation: Immunohistochemistry Histology and PolarizedLight Microscopy

1. Immunohistochemistry and Histology

Briefly, it is well known in the art that identification of bona fidearticular cartilage can be accomplished using ultrastructural and/orbiochemical parameters. For example, articular cartilage forms acontinuous layer of cartilage tissue possessing identifiable zones. Thesuperficial zone is characterized by chondrocytes having a flattenedmorphology and an extracellular network which does not stain, or stainspoorly, with toluidine blue, indicating the relative absence ofsulphated proteoglycans. Toluidine blue is commonly used for thestaining of bone and cartilage. It is a metachromatic stain that yieldsdifferent colors based on the presence of densely spaced negativecharges in the tissues leading to the aggregation and polymerization ofthe dye which shifts the color from blue to purple Bone is stained bluewhereas the cartilage, with its acidic mucopolysaccharides, is stained adark purple. Chondrocytes in the mid and deep zones have a sphericalappearance, and the matrix contains abundant sulphated proteoglycans, asevidenced by staining with toluidine blue. Collagen fibers are presentdiffusely throughout the matrix. The chondrocytes possess abundant roughendoplasmic reticulum and are surrounded by an extracellular network.The pericellular network contains numerous thin, non-banded collagenfibers. The collagen in the interterritorial network is less compactedand embedded in electron translucent amorphous material, similar toarticular cartilage. Collagen fibers in the interterritorial region ofthe network exhibit the periodic banding characteristic of collagenfibers in the interterritorial zone of cartilage tissue.

Von Kossa staining shows a dense black staining of the mineralizedtissue This stain clearly depicts the existing and newly regeneratedbone through the deposition of silver on the calcium salts. Typically,the counter stain is Safranin O, which stains the cartilage red-orange.New and existing bone can usually be easily distinguished formation.Exemplified herein are improved devices having parts of binding agent toparts of matrix ranging from approximately 1:1 to 4:1 up to andincluding at least 10:1, as well as from approximately 1:2 to 1:5, up toand including at least 1:10. Any proportion of binding agent to matrixcan be used to practice the instant invention. All that is required isadmixing binding agent with matrix and osteogenic protein so as toachieve bone and cartilage formation. As discussed below, certainbinding agents can be used in equal or greater proportions relative tomatrix, but such agents should be tested as taught herein to measure anymatrix dilution effects.

Those binding agents contemplated as useful herein include, but are notlimited to: art-recognized suspending agents, viscosity-producing agentsand emulsifying agents. In particular, art-recognized agents, such ascellulose gum derivatives and sodium alginate, gelatin powder anddextrans can be used. More particularly, cellulosic agents such asalkylcelluloses, including agents such as methylcellulose,methylhydroxyethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose, and hydroxyalkylcelluloses, to name but a few.Currently the most preferred is carboxymethylcellulose, including thesodium salt thereof. As exemplified below, other binding agents suitablefor use in the instant invention include, but are not limited to,dextran, mannitol, white petrolatum, sesame oil and admixtures thereof.In view of the teachings set forth herein, the artisan can identifysuitable equivalents of the above-identified binding agents using merelyroutine experimentation and ordinary skill. Suitable binding agentcandidates can be identified, characterized, tested and then used inosteogenic devices as set forth below.

In general, agents which are recognized in the art as suspending orviscosity-producing agents in pharmaceutical technologies are suitablefor use as a binding agent in the instant invention. Reference manualssuch as the USP XXII–NF XVII (The Nineteen Ninety U.S. Pharmacopeia andthe National Formulary (1990)) categorize and describe morphologicallyin sections stained accordingly. Safranin O/Fast Green is able todistinguish more features than the Toluidine blue. Safranin O is a basicdye that stains the acidic mucopolysaccharides in the articularcartilage red-orange and the underlying subchondral bone only lightly.Fast Green is an acidic dye that stains the cytoplasm gray-green. Stainis not only able to clearly identify the existing and regeneratedcartilage, but can also distinguish differences between two regions inthe reparative tissue indicating differences in the content ofproteoglycans.

Hematoxylin/eosin stains which depict bone a darker red and thecarbohydrate rich cartilage only very lightly, can also be used. MassonTrichrome is able to distinguish differences in the reparative tissue.Cartilage and acidic polysaccharide-rich reparative tissue, muscle, anderythrocytes are stained red, with the collagen of the bone stainedblue.

Histological evaluations can also involve assessment of:glycosaminoglycan content in the repair cartilage; cartilage andchondrocyte morphology; and, structural integrity and morphology at thedefect interface. The morphology of repair cartilage can be identifiedby the type of cartilage formed: articular vs. fibrotic by evaluatingglycosaminoglycan content, degree of cartilage deposition, and the like.

Histological evaluations using standard methodologies well characterizedin the art also allows assessment of new bone and bone marrow formation.See, for example, U.S. Pat. No. 5,266,683, the disclosure of which isincorporated herein by reference.

Additionally, it is well known in the art that biochemically, thepresence of Type II and Type IX collagen in the cartilage tissue isindicative of the differentiated phenotype of chondrocytes. The presenceof Type II and/or Type IX collagen can be determined by standard gelelectrophoresis, Western blot analysis and/or immunohisto-chemicalstaining using, for example, commercially available antibody asdescribed below. Other biochemical markers include hematoxylin, eosin,Goldner's Trichrome and Safranin-O.

Immunohistochemical methods, such as the following, can be utilized toidentify formation of cartilage tissue, including articular cartilage.Tissue sections are prepared using routine embedding and sectioningtechniques known in the art. Epitopes for Type II collagen are firstexposed by protease pretreatment. For example, tissue specimens arepretreated with 1 mg/ml pronase type XIV from Sigma (St. Louis, Mo.;catalog number P5147) in tris-buffered saline (TBS) for approximately 10min at room temperature. Specimens are then washed in TBS with 0.2%glycine. Specimens are blocked for 30 min, in a tris-buffered salinesolution containing 1% Tween 20 (TBST) and bovine serum albumin (BSA),and washed with TBST. Specimens are then incubated with affinitypurified polyclonal goat anti-human collagen Types I and II antibodiesfor approximately 1 hr, or overnight, at room temperature. In certain ofthe Examples set forth below, goat anti-human Type I collagen antibodywas obtained from Southern Biotechnology Associates (Birmingham,Alabama), catalog number 1310-01, for example, lot number L055-X916;goat anti-human Type II collagen antibody was also obtained fromSouthern Biotechnology Associates, catalog number 1320-01, for example,lot number C153-T826. Anti-human Types I and II collagen antibodiesgenerated in mouse or rabbit can also be used. The skilled artisan willappreciate the circumstances under which use of one species versusanother is appropriate. For certain of the examples set forth below, theconcentrations of goat anti-human Types I and II collagen antibodiesused for incubation is, for example, 20 μg/ml for each antibody dilutedinto 1% BSA in TBST. After incubation with antibodies, the specimens arerinsed with TBST and held in a bath. A commercially available linkantibody is then added. For example, specimens treated with goatanti-human collagen Types I and II antibodies can be incubated withgoat-link antibody from BioGenex Laboratories (San Ramon, Calif.);catalog number HK209-5G) for at least 10 min at room temperature. Forthose samples incubated with mouse or rabbit antibodies, a Dako LSAB2kit number K0610 from Dako Corporation (Carpinteria, Calif.) can be usedas the link antibody. The specimens are again rinsed with TBST and heldin a bath. Next, the specimens are allowed to incubate withStrepavidin/Alkaline Phosphatase commercially available from any of theabove-identified sources for at least approximately 10 min at roomtemperature. The specimens are again rinsed with TBST. The specimens arethen developed by treatment with an appropriate substrate solution forapproximately 10 min or less. For example, for alkaline phosphatasedetection, approximately 100 μl of 50× lavamesole is used. For colordevelopment, Fast Red from Dako Corporation is used. After development,the specimens are counterstained by washing for 2 min with Harrishematoxylin and 1% lithium carbonate. The specimens are then mounted inan aqueous mounting media, and cartilage formation is subsequentlyevaluated.

Staining for types I and II collagen is useful to determine the boundarybetween regenerated subchondral bone and reparative tissue. Generally,reparative tissue that is fibrous stains less intensely. Additionally,newly formed subchondral bone can be identified by type II collagenlocalization in small spicules of remnant cartilage. Toluidene blue andSafranin-O are also useful for staining acidic proteoglycans in acartilage layer as well as reparative tissues.

2. Polarized Light Microscopy

Polarized light microscopy can be used to assess fibril interdigitationat the junction between the margins of repair tissue and the residualarticular cartilage adjacent to the defect. Such microscopy can beperformed using Safranin-O stained sections from a defect. In certaininstances, polarized light microscopy offers the skilled artisan a moreaccurate view of the repair process. For example, using lightmicroscopy, repair tissue at the periphery of a defect can appear wellapposed with the residual cartilage. Using polarized light microscopy,however, it can be observed that the collagen fibrils of the repairtissue are not well integrated with those of the residual cartilage.Lack of fibril continuity between repair and persisting cartilage isindicative of sub-optimal repair. Thus, when evaluating qualitativelythe interface between repair cartilage and residual viable cartilage,fibrillar continuity is preferably assessed using polarized lightmicroscopy as exemplified herein below. (See, also, Shapiro et al.,Journal of Bone and Joint Surgery 75: 532–553 (1993), the disclosure ofwhich is herein incorporated by reference.)

V. ANIMAL STUDIES: METHODS OF USE OF IMPROVED OSTEOGENIC DEVICES

A. Repair of Critical Size Segmental Defects Using Improved OsteogenicDevices Containing Carboxymethylcellulose

1. Experiment 1 Unitary Device Configuration (Dogs)

This study illustrates the efficacy of OP-1 combined with collagenmatrix and carboxymethylcellulose for repairing critical-size ulnasegmental defects in the art-recognized canine model.

Briefly, the data set forth below indicate at least comparableradiographic healing at sites that received a CMC/OP-1 device relativeto segmental defects treated with the standard OP device. The finalradiographic grade (maximum=6.0) for defects treated with CMC/OP-1 was5.33±0.58 compared to 4.67±0.58 for defect receiving the standard OP-1device. In general, new bone formation was evident as early as two weekspost-operative in all defects. The new bone continued to densify,consolidate and remodel until sacrifice at twelve post-operative weeks.The mean load to failure of the defects treated with the CMC/OP-1 devicewas 59.33 N±26.77. This was 70% of the mean load to failure of thecontralateral sides which received the standard OP-1 implants.Histologically, the final volume, quality and degree of remodelling wereat least equivalent in defects treated with the CMC/OP-1 and standardOP-1 device, although a variation in the final new bone formation anddegree of remodelling was noted in animal to animal comparisons. Themean histologic grade for defects treated with the CMC/OP-1 device was12.67±1.04 out of 16 total possible points. The mean histologic gradefor defects treated with the standard OP-1 device was 11.41±0.95 out of16 total possible points.

Test Device Description

As already described, standard devices consisted of recombinant humanosteogenic protein-1 (rhOP-1) admixed with bovine bone Type I collagenmatrix at a ratio of 2.5 mg rhOP-1 per gram of collagen matrix. Theimproved device consisted of rhOP-1 admixed with bovine bone Type Icollagen matrix and carboxymethylcellulose (CMC). The unitary deviceswere supplied in sterile vials.

As earlier-described, the currently preferred CMC-containing device foropen defects has a putty consistency. The unitary CMC/OP-1 device wasplaced dry into a small bowl and mixed with saline. Using fingers, thepractitioner mixed and formed the device into the general shape of thedefect and then placed the device into the defect site. It was reportedthat the improved device was more easily handled and shaped, and did notstick to the surgical gloves. The device maintained its integrity whenplaced in the defect during irrigation and during/after suturing.

Experimental Design

Adult male mongrel dogs were utilized because of their well-known bonerepair and remodeling characteristics. All animals were at least twoyears old and weighed from 40 to 50 pounds. All animals were supplied byMartin Creek Kennels, USDA number 71-B-108, Willowford, Ark. Specialattention was paid in selecting animals of uniform size and weight tolimit the variability in bone geometry and loading. The animals wereradiographically screened pre-operatively to ensure proper size,skeletal maturity, and that no obvious osseous abnormalities existed.

A total of 3 adult male dogs were utilized. Bilateral 2.5 cm ulnasegmental defects were created. All right side defects received theimproved device (CMC/OP-1 device). All left side defects receivedstandard OP-1 device. Biweekly radiographs were taken to study theprogression of healing and graded on a 0–6 scale. At sacrifice, allulnae were retrieved en bloc, and those that were healed sufficientlyupon manual manipulation were mechanically tested in torsion. Segmentswere evaluated by histology for tissue response, bone architecture andremodelling, and quality and amount of new bone formation and healing;grading was on a 0–16 scale.

Surgery

Using standard aseptic techniques, surgery was performed under halothanegas anesthesia. A lateral incision approximately 4.0 cm in length wasmade and exposure of the ulna was obtained using blunt and sharpdissection. A 2.5 cm segmental osteoperiosteal defect was created in themid-ulna using an oscillating saw. This defect was about 2–2.5 times themid-shaft diameter, and represents a critical size defect, i.e., thedefect would not heal spontaneously. Intra-operative measurements weremade of the removed bone segment. The radius was maintained formechanical stability, but no internal or external fixation was used. Thesite was irrigated with saline to remove bone debris and spilled marrowcells. After the site was dried and homeostasis was achieved, theimplants were carefully placed into the defects. The soft-tissues weremeticulously closed in layers to contain the implant. The procedure wasthen repeated on the contralateral side.

Radiographs

Radiographs of the forelimbs were obtained biweekly until eight weekspost-operative and then again at sacrifice at twelve post-operativeweeks. Standardized exposure times and intensities were used, andsandbags were used to position the extremities in a consistent manner.Radiographs were evaluated and compared to earlier radiographs toappreciate quality and speed of defect healing. Grading of radiographswas in accordance with the following scale:

TABLE 5 Radiographic Grading Scale Grade: No change from immediatepost-operative appearance 0 Trace of radiodense material in defect 1Flocculent radiodensity with flecks of calcification 2 Defect bridged atleast one point with material of 3 non-uniform radiodensity Defectbridged on both medial and lateral sides with material 4 of uniformradiodensity, cut end of the cortex remain visible Same as grade 3; atleast one of four cortices is obscured by new 5 bone Defect bridged byuniform new bone; cut ends of cortex are no 6 longer distinguishable

Sacrifice

At the end of the study period, animals were sacrificed using anintravenous barbiturate overdose. The ulna and radius were immediatelyharvested en bloc and placed in saline soaked diapers. Both ulna weremacrophotographed and contact radiographs with labels were taken. Softtissues were carefully dissected away from the defect site. Awater-cooled saw was used to cut the ulna to a uniform length of 9 cmwith the defect site centered in the middle of the test specimen.

Mechanical Testing

Immediately after sectioning, if healing was deemed sufficient by manualmanipulation, specimens were tested to failure in torsion using routineprocedures on an MTS closed-loop hydraulic test machine (Minneapolis,Minn.) operated in stroke control at a constant displacement rate of 50mm/min. Briefly, each end of the bone segment was mounted in acylindrical aluminum sleeve and cemented with methylmethacrylate. Oneend was rigidly fixed and the other was rotated counterclockwise. Sincethe dog ulna has a slight curvature, the specimens were mounted to keepspecimen rotation coaxial with that of the testing device. The torsionalforce was applied with a lever arm of 6 cm, by a servohydraulicmaterials testing system. Simultaneous recordings were made of implantdisplacement, as measured by the machine stroke controller, while loadwas recorded from the load cell. Force-angular displacement curves weregenerated from which the torque and angular deformation to failure wereobtained, and the energy absorption to failure computed as the areaunder the load-displacement curve.

Histology

The individual specimens were fixed by immersion in 10% bufferedformalin solution immediately following mechanical testing or aftersectioning in untested specimens. On a water cooled diamond saw thespecimens were divided by bisecting the specimen down its long axis.This procedure, resulted in two portions of each specimen for differenthistologic preparations, including undecalcified ground sections andundecalcified microtome sections.

Following fixation, the specimens designated for undecalcified sectionswere dehydrated in graduated ethyl alcohol solutions from 70% to 100%.The specimens were then placed in methylmethacrylate monomer and allowedto polymerize. The ground sections were obtained by cutting thespecimens on a high speed, water cooled Mark V CS600-A (Grandby, Conn.)sectioning saw into sections approximately 700 to 1,000 μm thick.Sections were mounted on acrylic slides and ground to 100 μm thicknessusing a metallurgical grinding wheel, and microradiographs were madeusing standardized techniques. Following microradiography, the sectionswere further ground to approximately 50 μm and stained with basicfuchsin and toluidine blue for histologic grading that evaluated thefollowing parameters of repair: quality of the union, the appearance andquality of the cortical and cancellous bone, the presence of bone marrowelements, bone remodelling, and inflammatory response. Grading ofhistologic parameters was in accordance with the following scale:

TABLE 6 Histologic Grading Scale Grade: Quality of Union: no sign offibrous or other union 0 fibrous union 1 osteochondral union 2 boneunion 3 bone union with reorganization of cortices 4 Cortex Development:none present in the defect 0 densification of borders 1 recognizableformation 2 intact cortices but not complete 3 complete formation ofnormal cortices 4 Residual Implant Material/Internal Architecture: largeamounts of implant material visible 0 moderate amount of residualimplant material 1 small amount of residual implant 2material/unorganized architecture no residual implant/return of marrow 3cavity/some marrow elements normal marrow elements and architecture 4Inflammatory Response: severe 0 severe/moderate 1 moderate response 2mild 3 no response 4 TOTAL POINTS 16 

Results Radiographic Evaluation

In this study, there were no significant differences in the radiographicbone healing characteristics of sites that received a CMC/OP-1 device ascompared to segmental defects treated with the standard OP device. Ingeneral, new bone formation was evident as early as two weekspost-operative in all defects. The new bone continued to densify,consolidate and remodel until sacrifice at twelve post-operative weeks.New cortex development with early medullary cavity formation occurredbetween the 6 and 8 week evaluations. The final radiographic grade fordefects treated with the CMC/OP-1 device was 5.33±0.58. The finalradiographic grade for defects receiving the standard OP-1 device was4.67±0.58.

As an example, specific representative observations for one of the testanimals are set forth below:

Right Defect (CMC/OP-1 Device)

At two weeks post-operative, traces of radiodense material were presentin the right defect, but, the defect was not completely bridged orfilled with new bone. By four weeks post-operative, the amount andradiodensity of the new bone significantly increased. The defect wasspanned, but, the new bone was not well contained. There was someconsolidation of new bone along the periosteal borders. An equivalentamount of new bone had formed compared to the left defect of thisanimal. At six weeks post-operative, the radiodensity of the new boneincreased and the defect was completely spanned and filled withextensive new bone. Early remodelling was evident with the host boneends beginning to incorporate with the new bone. At eight weekspost-operative, new bone continued to remodel and the new bone volumebetter approximated the defect borders. The host bone ends wereincorporated with the new bone with densification of new bone along theborders suggestive of new cortex formation. There was no radiographicevidence of any residual carrier material. At sacrifice a radiolucentregion was present within the center of the right side defect, but,densification of the new bone borders was suggestive of new cortexformation. The final radiographic grade was 5 out of 6 possible points.

Left Defect (OP-1 Device)

At two weeks post-operative, traces of radiodense material were presentin the defects, but, the defect was not bridged or filled with new bone.At four weeks post-operative, the amount and radiodensity of the newbone significantly increased and the defect was spanned and filled withnew bone. At six weeks post-operative, the radiodensity of the new boneincreased and the defect was completely spanned and filled withextensive new bone. Early remodelling was evident, and the new bonevolume better approximated the defect borders. The new bone wasuniformly dense, and the host bone ends were beginning to incorporate.At eight weeks post-operative new bone continued to remodel, the hostbone ends were incorporated, and densification of the new bone had begunalong the defect borders. At sacrifice densification of the new bonealong the defect borders was suggestive of early reformation of newcortices. The density of the new bone within the center of the defectwas greater than new bone that had formed in the right side defect,although there were no significant differences in the radiographicappearances of the right and left sides in this animal. The finalradiographic grade was 5 out of 6 possible points.

Gross Observations

Both right and left specimens from all animals had similar grossappearances. In two animals, the right and left defects were firmlyunited and had approximately the same volume of new bone. In a thirdanimal, both the right and left side had a similar new bone volume, but,the left side was not completely united.

Mechanical Testing

The mean load to failure for defects treated with the CMC/OP-1 devicewas 59.33 N±26.77 (n=3). The mean load to failure was 79% of the meanload to failure of the contralateral sides, which received the standardOP-1 devices. This represented 91% of the strength of intact controlstested previously. The mean angular deformation was 38.22±0.69 degrees.The mean energy absorbed to failure was 97.47±47.21 Nm degrees.

The mean load to failure of the defects treated with the standard OP-1device was 75.39 N±1.88 (n=2). This represented 115% of the strength ofintact controls tested previously. The mean angular deformation was59.06±27.80 degrees. The mean energy absorbed to failure was 93.40±17.49Nm degrees. As noted, one defect treated with the standard OP-1 devicewas not tested due to gross instability.

Histology

In general, normal bone formation consistent with defects treated withthe standard rhOP-1 device with a collagen matrix were observed. Bothfinal volume and quality and degree of remodelling were equivalent incomparison of CMC/OP-1 and standard OP-1 devices. A variation in thefinal new bone formation and degree of remodelling was noted in animalto animal comparisons. The mean histologic grade for defects treatedwith the CMC/OP-1 device was 12.67±1.04 out of 16 total possible points.The mean histologic grade for defects treated with the standard OP-1device was 11.41±0.95 out of a total of 16 possible points.

Generally, both the left and right defects were spanned by a largevolume of new bone. The new bone was beginning to reorganize and hadlamellar characteristics. Along the defect borders, the new bone becamemore dense and was suggestive of new cornices. In certain instances,remodelling was not as uniformly advanced on the left as the right side.The volume of new bone on the left side was slightly less than on theright in certain instances. At the center of all the defects, the returnof medullary components was evident.

In conclusion, improved osteogenic devices (unitary configuration) wereused to repair critical size segmental defects. Rates of endochondralbone repair, mechanical strength indicia, radiographic indicia andhistological indicia suggested improved devices result in defect repairat least comparable to standard osteogenic devices.

2. Experiment 2 OP-1 Dose Response Using Non-unitary DeviceConfiguration (Dogs)

This study further illustrates the efficacy of standard osteogenicdevice admixed with carboxymethylcellulose (CMC) using both standard andlow OP-1 dose formulations to heal large, critical size segmentaldefects in the canine ulna segmental defect model.

As described in detail below, various dosages of OP-1 were employed inthis study. Briefly, the low dose formulations of the OP-1 devicewithout CMC were found effective at inducing new bone formation, butless so than the standard dose OP-1 device. However, and unexpectedly,defects treated with the low dose CMC-containing device demonstratedearlier and larger volumes of new bone formation compared to the lowdose OP-1 device without CMC. The standard or low dose OP-1 device wasprepared by combining a 1 g OP-1 device with 3.2 ml sterile saline. Thestandard or low dose OP-1 device containing CMC was prepared bycombining 1 g OP-1 device with 0.2 g CMC and approximately 2 ml sterilesaline. The devices were prepared intra-operatively. Radiographically,standard dose OP-1 treated sites with and without CMC had similarradiographic appearances. Standard dose OP-1 sites had earlier andgreater volumes of new bone formation compared to the low dose sites.Histologic results demonstrated more advanced segmental bone defecthealing in sites treated with the CMC-containing device compared to thestandard OP-1 device. Sites treated with low dose OP-1 device containingCMC achieved an equivalent degree of remodeling and incorporation withthe host bone relative to sites treated with the standard dose OP-1device, but, the volume of new bone induced was less. Defect sitestreated with standard dose device containing CMC obtained the greatestmean torsional load to failure at twelve weeks post-operative comparedto all other treatment groups (61.91±35.37 N, 95% of the torsionalstrength of intact controls). The torsional strength of the low dosedevice containing CMC sites was similar to the standard OP-1 device,having 78% of the strength of intact ulnae and 99% of the strength ofpreviously tested sites treated with the standard device. In contrast,the torsional strength of the low dose OP-1 sites without CMC was only44% of the torsional strength of intact ulnae and 56% of previouslytested segmental defects treated with the standard OP-1 device.

Test Material

The standard OP-1 device (designated OP in Table 11) consisted ofrecombinant human osteogenic protein-1 (rhOP-1) admixed with bovine boneType I collagen matrix at a ratio of 2.5 mg rhOP-1/g of collagen matrix.One CMC device (standard dose, designated OP-1/CMC, OPCMC in Table 11)consisted of an OP-1 device combined with carboxymethylcellulose. Thelow dose OP-1 device consisted of 1.25 mg rhOP-1/g of collagen matrix(designated LOP in Table 10). Each OP-1 device in both standard and lowdose consisted of 1 g of device packaged separately from CMC. CMC waspackaged 200 mg per vial. This is in contrast to the unitary devicedescribed above wherein CMC was co-packaged with the other components ofcollagen matrix and osteogenic protein.

Experimental Design

A total of 12 adult mongrel dogs were utilized. Bilateral 2.5 cmcritical size ulna segmental defects were created. The right sidedefects of six animals received the standard OP-1 device. The left sidedefects of this group received the standard dose OP-1/CMC devices. Thesecond group of six animals received the low dose OP-1 devices in theright side defect and received low dose OP-1/CMC devices in the leftside defects. Biweekly radiographs were taken to study the progressionof healing. At sacrifice, all were retrieved in bloc and mechanicallytested in torsion. Ulna segments were evaluated by histology for tissueresponse, residual implant, and quality and amount of new bone formationand healing.

Animal Model

As described above, adult male mongrel dogs were utilized because oftheir anatomical size, and known bone repair and remodelingcharacteristics. All animals were skeletally mature and weighed from 35to 50 pounds.

Surgery

Using standard surgical techniques similar to those described above, alateral incision approximately 4 cm in length was made and exposure ofthe ulna was obtained using blunt and sharp dissection. A 2.5 cmsegmental osteoperiosteal defect was created in the mid-ulna using anoscillating saw. This defect was about 2–2.5 times the mid-shaftdiameter, and represented a critical-sized defect, i.e., the defectwould not heal spontaneously. Intra-operative measurements were made ofthe removed bone segment. The length of the segment, the two outerdiameters of the segment, and the central diameter of the segment wasrecorded in millimeters in the surgical records. The radius wasmaintained for mechanical stability. The site was irrigated with salineto remove bone debris and spilled marrow cells. After the site was driedand homeostasis was achieved, the implants were placed in the defect.The soft-tissues were closed in layers to contain the implant. Theprocedure was then repeated on the contralateral side.

Radiographs

As described above, radiographs of the forelimbs were obtained biweeklyuntil eight weeks post-operative and then again at sacrifice at twelvepost-operative weeks.

Sacrifice

Procedures were similar to these described above. At the end of thestudy period, animals were sacrificed, the ulna and radius immediatelyharvested en bloc and placed in saline soaked diapers. Soft tissues werecarefully dissected away from the defect site. A band saw was used tocut the ulna to a uniform length of 9 cm with the defect site centeredin the middle of the test specimen.

Mechanical Testing

Protocols were similar to those described above. Briefly, specimens weretested to failure in torsion on an MTS closed-loop hydraulic testmachine (Minneapolis, Minn.) operated in stroke control at a constantdisplacement rate of 50 mm/min. Torsional force was applied with a leverarm of 6 cm, by a servohydraulic materials testing system. Simultaneousrecordings were made of implant displacement, as measured by the machinestroke controller, while load was recorded from the load cell.Force-angular displacement curves were generated from which the torqueand angular deformation to failure were obtained, and the energyabsorption to failure was computed as the area under theload-displacement curve.

Histology

As described above, fixed specimens designated for undecalcifiedsections were dehydrated in graduated ethyl alcohol solutions from 70%to 100%. The specimens were then placed in methylmethacrylate monomerand allowed to polymerize. The ground sections were obtained by cuttingthe specimens on a high speed, water cooled sectioning saw into sectionsapproximately 700 to 1,000 μm thick. These sections were mounted onacrylic slides and ground to 100 μm thickness. Following routinemicroradiography, the sections were further ground to approximately 50μm and stained with basic fuchsin and toluidine blue for histologicgrading that evaluated the following parameters of repair: quality ofthe union, the appearance and quality of the cortical and cancellousbone, the presence of bone marrow elements, bone remodeling, andinflammatory response.

Radiographic Evaluation

At twelve weeks post-operative (sacrifice), the standard dose OP-1/CMCsites achieved the greatest mean radiographic grade, 5.17/6.0 points.The final radiographic grade for the standard OP-1 devices was 5.00/6.0.The low dose OP-1 sites had a mean final radiographic grade of 3.83/6.0.Low dose OP-1/CMC sites had a mean grade of 4.67/6.0. At all timeperiods the standard dose OP-1/CMC sites had greater mean radiographicgrades than standard OP-1 without CMC. At all time periods the low doseOP-1 CMC sites had greater mean radiographic grades than low dose OP-1without CMC sites.

Statistical analysis demonstrated a significant effect for implant typewhen all radiographic grades were combined (Kruskal-Wallis one wayanalysis of variance, p=0.0049). Multiple comparisons demonstrated thatthe standard OP-1 and the standard dose OP-1/CMC devices meanradiographic grades for all time periods were significantly greater thanthe low dose OP-1 sites without CMC (at α=0.10 and at α=0.05,respectively). Multiple comparisons also demonstrated that the meanradiographic grade of the standard dose OP-1/CMC sites was significantlygreater than the low dose OP-1/CMC sites (α=0.10). However, andunexpectedly, the standard dose OP-1 devices mean radiographic gradeswere not significantly greater than the mean radiographic grades for thelow dose OP-1/CMC sites.

Low Dose OP-1 Sites without CMC

At two weeks post-operative, new bone formation was evident in one ofsix defects treated with low dose OP-1. Traces of radiodense materialwere present around the defect, but new bone did not bridge or span thedefect. The mean radiographic grade at two week post-operative was0.17/6.0 points. At four weeks post-operative, the same sitedemonstrating new bone formation at two weeks demonstrated an increasein new bone volume. Four defects were spanned and one defect was filledwith new bone at four weeks. Two sites demonstrated little activity atfour weeks post-operative. The mean radiographic grade at four weeks was1.83/6.0. By six weeks post-operative, two sites treated with low doseOP-1 were spanned and filled with new bone. Two sites were spanned butincompletely filled with new bone. One animal demonstrated some earlynew bone formation. One animal did not demonstrate any new boneformation. The mean radiographic grade at six weeks was 2.83/6.0. Fromsix to eight weeks additional new bone formation was not evident, but,some densification of new bone was apparent and some early remodelinghad occurred. The mean radiographic grade at eight weeks was 3.17/6.0.At sacrifice, twelve weeks post-operative, all defects demonstrated somewell contained new bone, but, the density was significantly less thanthe surrounding host bone. Occasional radiolucencies at the host bonenew bone junction were present. The mean radiographic grade at twelveweeks was 3.83/6.0.

Low Dose OP-1/CMC Sites

At two weeks post-operative, early new bone formation was evident inthree of six defects treated with low dose OP-1/CMC. New bone did notspan or fill the defects, but was well contained within the surgicalsites. The mean radiographic grade at two weeks was 0.83/6.0. At fourweeks post-operative, new bone formation was present in five of sixdefects, spanning and almost filling the defects. The mean radiographicgrade at four weeks was 2.33/6.0. At six weeks the density of new bonepresent in the defects increased. Early incorporation of the host bonewas evident in three of six defects. One animal did not demonstrate anynew bone formation bilaterally at six weeks. The mean radiographic gradeat this time was 3.00/6.0. From six to eight weeks, no additional newbone formation occurred. Early remodeling and incorporation with thehost bone was apparent. One animal did not demonstrate any changes inradiographic appearance. The mean radiographic grade at eight weeks was3.33/6.0. Unexpectedly, the low dose OP-1/CMC sites demonstrated moreextensive new bone formation and remodeling than the low dose OP-1 siteswithout CMC. In sites where the defect was completely filled with newbone, the density of the new bone was less than the surrounding hostbone. The mean radiographic grade at twelve weeks (sacrifice) was4.67/6.0.

Standard OP-1 Device Sites

The results in this study were consistent will all previous studies ofthe standard OP-1 device. At two weeks post-operative, four of sixdefects treated with the OP-1 device demonstrated early new boneformation. In two defects, extensive new bone spanned the defects, but,new bone did not fill the defects. Overall, the new bone was not wellcontained. The mean radiographic grade at two weeks was 1.50/6.0. Atfour weeks post-operative, an increase in the amount and density of newbone occurred in all six defects. New bone spanned all defects. In fourof six sites the defect appeared completely filled with new bone. Themean radiographic grade at four weeks was 3.00/6.0. At six weekspost-operative, the density of new bone increased. New bone was not wellcontained in the remaining defects. Generally, early incorporation atthe host bone ends was observed in three of the six sites. The meanradiographic grade at six weeks was 3.67/6.0. From six to eight weeks,nearly complete incorporation with the host bone was evident in three ofsix sites, although remodeling toward the ulna contours had occurred inall defects. The mean radiographic grade at eight weeks was 4.50/6.0. Bytwelve weeks post-operative (sacrifice) extensive remodeling hadoccurred, although the new bone volume did not yet approximate the ulnacontours. New bone often extended into the surrounding soft tissues,although some reformation of cortices was apparent in all defectstreated with the standard OP-1 device. The mean radiographic grade attwelve weeks was 5.00/6.0.

Standard Dose OP-1/CMC Sites

At two weeks post-operative, early new bone formation was evident infour of six defects treated with OP-1/CMC device. New bone was wellcontained in only one of the four defects. New bone appeared to span andfill two of the six defects. The mean radiographic grade at two weekswas 1.67/6.0. At four weeks post-operative, extensive new bone hadoccurred in all six defects. New bone was not well contained, but, earlyincorporation with the host bone was observed in two sites. The meanradiographic grade at four weeks was 1.67/6.0. From four to six weeks,extensive remodeling and incorporation of the host bone occurred in alldefects. New bone was not well contained, but, new bone in the softtissue had begun to resorb. The mean radiographic grade at six weeks was4.33/6.0. By eight weeks, complete incorporation with the host bone wasappreciated in two sites, and early new cortex formation was evident inat least one site. The mean radiographic grade at eight weeks was4.67/6.0. At twelve weeks post-operative (sacrifice), three of sixdefects had extensive incorporation with the host bone ends. New bonewas still present outside of the defects, although extensive remodelinghad occurred. The mean radiographic grade at twelve weeks was 5.17/6.0.

Gross Observations

Sites treated with low dose implants with and without CMC demonstratedless new bone volume compared to the high dose OP-1 sites with andwithout CMC. All high dose sites were firmly united grossly, but threeof twelve sites treated with low dose OP-1 were not yet firmly united atsacrifice.

Low Dose OP-1 Sites without CMC

In all cases, the amount of new bone formed did not exceed the originaldefect volume. New bone formation was well contained, although in two ofsix segments the bone was not completely united.

Low Dose OP-1/CMC Sites

Similar to defects treated with low dose OP-1, new bone formation waswell contained. One of six sites treated with low dose OP-1/CMC was notcompletely united. Typically, new bone volume was less than the originaldefect volume.

Standard OP-1 Device Sites

Similar to previous studies, new bone volume in sites treated with thestandard OP-1 device was 2 to 3 times greater than the original defectvolume. All defects were firmly united. In five of six defects,extensive new bone extended into the soft tissues and was fused to theradius. In one defect, the volume of new bone formed was less than othersites.

Standard Dose OP-1/CMC Sites

New bone volume in five of six defects treated with OP-1/CMC exceededthe original host bone volume and extended into the soft tissues. Thenew bone volume was 2 to 3 times the volume of the original defect. Asnoted above in one animal, reduced bone volume was observed bilaterally.

Mechanical Testing

Mechanical testing summaries appear in Tables 7 and 8.

Unexpectedly, defect sites treated with the standard dose OP-1/CMCdevice obtained the greatest mean torsional load to failure at twelveweeks post-operative compared to all other treatment groups, includingthe standard device group. The mean load to failure was 61.91±35.37 N(n=6). This represented 95% of the torsional strength of previouslytested intact ulnae and 121% of the strength of previously testedsegmental defects treated with the standard OP-1 device. The standardOP-1 device treated sites had a mean torsional strength of 55.84±37.26 N(n=6), 86% of previously tested intact control ulnae, and 110% ofpreviously tested segmental defects treated with the OP-1 device. Themean load to failure for the low dose OP-1/C MC sites was 50.66±31.68 N(n=5), or 78% of the strength of intact control ulnae and 99% of thestrength of previously tested segmental defects treated with thestandard OP-1 device. The mean load to failure for the low dose OP-1sites was 28.72±14.71 N (n=4). This represented 44% of the torsionalstrength of previously tested intact control ulnae and 56% of previouslytested segmental defects treated with the standard OP-1 device.

Unexpectedly, paired t-tests of the failure load within animalsdemonstrated a significant effect for implant type when comparing lowdose OP-1 standard devices to low dose OP-1/CMC devices (p=0.0597). Thepaired mean load for low dose OP-1 sites was 28.72±14.71 (4). The pairedmean load to failure for the low dose OP-1/CMC sites was 62.89±18.47(4). No significant difference was found in paired t-tests of mean loadto failure standard OP-1 devices compared to the mean load to failurestandard dose OP-1/CMC device.

TABLE 7 Mechanical Testing Results Energy absorbed Torque % IntactControls Angulation to failure Animal Number side Implant Type Load toFailure (Nm) (%) (degrees) (NmDegrees) H19 Right LOP 35.17 2.11 53.8836.74 53.93 H21 Right LOP 9.76 0.59 14.95 66.64 16.96 H24 RightLOP * * * * H26 Right LOP 25.75 1.55 39.45 41.33 44.42 H30 RightLOP * * * H34 Right LOP 44.18 2.65 67.69 25.35 41.51 MEAN 28.72 1.7243.99 42.52 39.21 STANDARD DEVIATION 14.71 0.88 22.53 17.43 15.75 SAMPLESIZE 4 4 4 4 4 H19 Left LOPCMC 39.76 2.39 60.92 37.13 55.84 H21 LeftLOPCMC 57.51 3.45 88.11 52.47 59.47 H24 Left LOPCMC 1.74 0.10 2.67 5.540.34 H26 Left LOPCMC 82.33 4.94 126.14 37.85 120.58 H30 LeftLOPCMC * * * * H34 Left LOPCMC 71.94 4.32 110.22 42.47 127.67 MEAN 50.663.04 77.61 35.09 72.78 STANDARD DEVIATION 31.68 1.90 48.54 17.62 52.46SAMPLE SIZE 5 5 5 5 5

TABLE 8 Mechanical Testing Results Energy absorbed Torque % IntactControls Angulation to failure Animal Number side Implant Type Load toFailure (Nm) (%) (degrees) (NmDegrees) H22 Right OP 65.44 3.93 100.2652.95 139.25 H23 Right OP 2.73 0.16 4.18 15.90 8.91 H25 Right OP 52.783.17 80.86 31.36 70.65 H28 Right OP 27.24 1.63 41.73 53.04 49.49 H32Right OP 79.68 4.78 122.08 19.33 57.83 H33 Right OP 107.14 6.43 164.1529.3 98.59 MEAN 55.84 3.35 85.54 33.65 70.79 STANDARD DEVIATION 37.262.24 57.08 16.08 44.52 SAMPLE SIZE 6 6 6 6 6 H22 Left OPCMC 76.52 4.59117.24 44.86 150.86 H23 Left OPCMC 6.53 0.39 10.10 68.10 10.66 H25 LeftOPCMC 50.22 3.01 76.94 43.94 69.48 H28 Left OPCMC 100.44 6.03 153.8840.9 177.13 H32 Left OPCMC 43.82 2.63 67.14 40.2 70.58 H33 Left OPCMG93.93 5.64 143.91 43.96 156.09 MEAN 61.91 3.71 94.85 46.99 105.80STANDARD DEVIATION 35.37 2.12 54.19 10.51 65.21 SAMPLE SIZE 6 6 6 5 6*Specimen was not tested

Histology

Unexpectedly, the sites treated with the standard dose OP-1/CMC deviceachieved the greatest mean histologic score, 12.08/16.0 points. The lowdoes OP-1 CMC sites achieved a score of 11.07/15.0, slightly greaterthan the mean histologic score for the standard OP-1 device sites,10.88/16.0. The mean histologic grade for the low does OP-1 sites was9.58/16.0 points.

Statistical analysis of the mean histologic grades by treatment groupdemonstrated a significant effect for implant type (Kruskal-Wallis oneway analysis of variance, p=0.0282). Multiple comparisons of group meansdemonstrated that the mean total grade for the standard dose OP-1/CMCsites was significantly greater than the low dose OP-1 without CMC sites(at α=0.05).

Statistical analysis of the grade for quality of union also demonstrateda significant effect for implant type. Unexpectedly, the mean quality ofunion grade for the standard dose OP-1/CMC sites (3.5/4.0) was againsignificantly greater than the low dose OP-1 sites (2.0/4.0, at α=0.05).No significant differences were found for implant type when comparingmean grades for cortex development, residual implant, and inflammatoryresponse.

Low Dose OP-1 Sites without CMC

New bone formation was apparent in all defects treated with low doseOP-1, but the amount of new bone within the defect often did not fillthe defect and was not continuous with the host bone ends. In one sitethe defect completely united histologically. New bone was in the earlystages of organization and remodeling. Some areas of newly mineralizingbone were also evident.

Low Dose OP-1/CMC Sites

The low dose OP-1/CMC sites had a similar histologic appearance comparedto the low dose OP-1 sites. However, and unexpectedly, new bone wascontinuous with the host bone more frequently in the low dose OP-1/CMCsites compared to the low dose OP-1 sites. In cases where the bone wascontinuous with the host bone, early remodeling and densification of thenew bone borders was apparent. In cases where new bone healing was notcomplete, areas of newly mineralizing bone were apparent, as well asareas of fibrous tissues within the defect. In general, the new bone waswell contained. Some areas of advanced remodeling along the defectborders was observed.

Standard OP-1 Device Sites

Extensive new bone formation bridged all defects. Early densification ofthe new bone borders had occurred. In some cases, areas of newlymineralizing bone joined areas of mature bone. At the center of thedefects, occasional small areas of residual carrier material waspresent. No inflammatory response was observed. New bone often extendedinto the soft tissues. Remodeling was most advanced at the defect/newbone borders. The bone had remodeled to a lamellar structure in theseareas.

Standard Dose OP-1/CMC Sites

There were no marked differences in the histologic appearance betweenthe standard OP-1 sites and the standard dose OP-1/CMC sites. Extensivenew bone spanned and filled the defects. The most extensive remodelingoccurred at the new bone/host bone borders. The remodeled bone had alamellar structure in these areas. Densification of new cortices wasevident, but not yet complete. Occasional small amounts of trappedresidual carrier material surrounded by new bone formation wereobserved. There was no associated, inflammatory response.

Conclusion

Improved osteogenic devices unexpectedly induced earlier and largervolumes of new bone formation at low doses of OP-1 than were induced bystandard devices at low doses of OP-1. Moreover, and unexpectedly,defect sites treated with improved osteogenic devices achieved thegreatest mean torsional load to failure at twelve-weeks post-operative.Histologically, improved devices unexpectedly achieved the greatest meanscore and more frequently demonstrated continuous new bone with hostbone.

B. Repair of Non-Critical Size Segmental Defects Using ImprovedOsteogenic Devices Containing Carboxymethylcellulose

1. Experiment 1 Time Course of Repair of Closed Defect as Treated with aUnitary Device (Dogs)

This non-critical size gap study was conducted to evaluate injectableconfigurations of improved osteogenic devices. The study design used the3 mm gap at 4 week model. The study evaluated the healing of the defectafter injection of OP-1/CMC/collagen matrix configuration. Thecontralateral arm of each animal was a control. In addition, a healingtime course for an untreated defect was evaluated at 4, 8 and 12 weeks.

The details of the protocol used are summarized below.

Test System

Adult mongrel dogs (18) bred for purpose were utilized in this studybecause of their anatomical size and known bone repair and remodelingcharacteristics. The animals were approximately 2 to 4 years old atonset of study and weighed 20 to 30 kg (approximately). The animals wereradiographically screened to ensure proper size, skeletal maturity, andthat no obvious osseous abnormalities exist.

Test Material Description

Improved osteogenic device formulations comprising recombinant humanosteogenic protein-1 (rhOP-1) in a collagen matrix admixed with CMC weretested. Controls consisted of mock device alone.

-   -   Formulation 1: 0.350 mg rhOP-1 in 100 μl CMC gel (7%) w/o        collagen matrix    -   Formulation 2: 0.350 mg rhOP-1 in 100 μl acetate/lactose buffer    -   Formulation 3: 0.350 mg rhOP-1 in 170 mg collagen-CMC matrix        wetted with saline    -   Control 1: 0 mg rhOP-1 in 100 μl gel    -   Control 2: 0 mg rhOP-1 in 100 μl acetate/lactose buffer    -   Control 3: 0 mg rhOP-1 in 170 mg collagen-CMC matrix wetted with        saline

Experimental Design

Bilateral 3 mm ulna segmental defects were created in all animals. Nineanimals received one of the three experimental test formulations in theright side defect, such that three sites of each type were studied. Theleft defect was implanted with mock device. These animals weresacrificed at four weeks post-operative. The remaining nine animalsreceived non-implanted defects bilaterally and were sacrificed atperiods at four, eight, and 12 weeks (three at each time period). Asdiscussed above, radiographs were taken to study the progression ofhealing. Final determination of sacrifice dates of the nine animalsreceiving rhOP-1 formulations was based upon the weekly radiographs. Atsacrifice, all ulnae were retrieved en bloc and mechanically tested intorsion. Segments were evaluated by histology, as described above, fortissue response, and quality and amount of new bone formation, andextent of healing.

Using standard surgical techniques, a lateral incision approximately twocentimeters in length was made, and exposure of the ulna was obtainedusing blunt and sharp dissection. The 3 mm defect was created in theright mid-ulna using an oscillating saw. The radius was maintained formechanical stability, but no internal or external fixation was used. Thesoft-tissues were meticulously closed in layers around the defect. TherhOP-1 sample or mock device was then injected into the site as per thetreatment schedule. The procedure was then repeated on the contralateralside with the appropriate sample.

Radiographs of the forelimbs were obtained weekly until six weekspost-operative and then biweekly until 12 weeks in the survivinganimals. One additional x-ray was obtained from the remaining animals atsacrifice at twelve weeks post-operative. Radiographs were graded by theinvestigator on a 0–6 grading scale and compared to earlier radiographsto appreciate quality and speed of defeat healing.

Testing Procedures

As discussed above, the animals were sacrificed at the designated times,and the ulna and radius were immediately harvested en bloc. Both ulnawere macrophotographed and contact radiographs taken. Soft tissues weremeticulously dissected away from the defect site. A water-cooled saw wasused to cut the ulna to a uniform length of 9 cm with the defect sitecentered in the middle of the test specimen. Immediately aftersectioning, the specimen was tested in torsion to failure on an MTSclosed-loop hydraulic test machine (Minneapolis, Minn.), as describedabove.

Both tested and untested specimens were prepared for histologicevaluation, as already described above. Following microradiography, thesections were further ground to approximately 50 μm and stained withbasic fuchsin and toluidine blue for histologic evaluation of parametersof repair including: the quality of the union, the appearance andquality of the cortical and cancellous bone, and the inflammatoryresponse.

Descriptive statistics of mechanical testing, radiographic grading andhistology were evaluated to characterize healing.

Results

The following observations and representative data were collected todate (4 weeks post-operative):

Mechanical testing summaries appear in Tables 9, 10 and 11. Table 11 isa summary of control subjects in previous, unrelated experiments.Generally and overall, the results of this study indicate that animalstreated with OP-1 exhibit accelerated healing. The OP-1 treated defectshealed in one-third to one-half the time of untreated controls.Additionally, and unexpectedly, the CMC/OP-1/collagen formation resultedin better bone containment than observed in the absence of CMC. Theseobservations were confirmed mechanically, radiographically andhistologically.

Conclusion

CMC-containing osteogenic devices (injectable configuration) can be usedto repair non-critical size, 3 mm ulna segmental defects at a closeddefect site.

TABLE 9 Evaluation of Improved Device Formulations of rhOP-1 for Repairof Noncritical Size Defects MECHANICAL TESTING RESULTS Percent intactEnergy absorbed Maximum Load to Failure Torque control to failure AnimalNumber Side Time Period Implant (N) (Nm) (%) Angulation (Nm-degrees)18750 right 4 weeks FORMULATION 1 49.37 2.96 75.65 35.72 63.57 18643right 4 weeks FORMULATION 1 16.56 0.99 25.37 14.04 6.78 18043 right 4weeks FORMULATION 1 33.32 2.00 51.05 43.62 54.56 MEAN 33.08 1.99 50.6931.13 41.64 STANDARD 16.41 0.98 25.14 15.32 30.52 DEVIATION SAMPLE SIZE3 3 3 3 3  17884* right 4 weeks FORMULATION 2 32.47 1.95 49.75 50.1155.91 18473 right 4 weeks FORMULATION 2 43.83 2.63 67.15 37.53 51.77right 4 weeks FORMULATION 2 10.79 0.65 16.53 20.78 5.94 MEAN 20.03 1.7444.48 36.14 37.87 STANDARD 16.79 1.01 25.72 14.71 27.73 DEVIATION SAMPLESIZE 3 3 3 3 3 18772 right 4 weeks FORMULATION 3 42.64 2.56 65.33 55.0655.74 18640 right 4 weeks FORMULATION 3 20.95 1.26 32.10 24.77 14.6218508 right 4 weeks FORMULATION 3 7.24 0.43 11.09 17.08 3.04 MEAN 23.611.42 36.17 32.30 24.47 STANDARD 17.85 1.07 27.35 20.08 27.70 DEVIATIONSAMPLE SIZE 3 3 3 3 3 *Calculations based on raw data from mechanicaltesting printout.

TABLE 10 Evaluation of Improved Device Formulations of rhOP-1 for Repairof Noncritical Size Defects MECHANICAL TESTING RESULTS Percent intactEnergy absorbed Maximum Load to Failure Torque control to failure AnimalNumber Side Time Period Implant (N) (Nm) (%) Angulation (Nm-degress)18750 left 4 weeks CONTROL 1 12.81 0.77 19.63 33.26 14.06 18643 left 4weeks CONTROL 1 11.00 8.00 16.85 59.35 16.83 18043 left 4 weeks CONTROL1 4.14 0.25 6.34 7.46 0.70 MEAN 9.32 3.01 14.27 33.36 10.53 STANDARD4.57 4.33 7.01 25.95 8.62 DEVIATION SAMPLE SIZE 3 3 3 3 3  17884* left 4weeks CONTROL 2 4.82 0.29 7.38 11.12 0.73 18473 left 4 weeks CONTROL 24.53 0.27 6.94 30.63 2.50 left 4 weeks CONTROL 2 7.52 0.45 11.52 33.298.59 MEAN 5.62 0.34 8.62 24.91 3.94 STANDARD 1.65 0.10 2.53 12.03 4.12DEVIATION SAMPLE SIZE 3 3 3 3 3 18772 left 4 weeks CONTROL 3 15.41 0.9223.61 49.91 15.40 18640 left 4 weeks CONTROL 3 10.28 0.62 15.75 40.5011.23 18508 left 4 weeks CONTROL 3 4.32 0.26 6.62 4.89 0.41 MEAN 10.000.60 15.33 31.77 9.01 STANDARD 5.55 0.33 8.50 23.75 7.74 DEVIATIONSAMPLE SIZE 3 3 3 3 3 *Calculations based on raw data from mechanicaltesting printout.

TABLE 11 Evaluation of Improved Device Formulations of rhOP-1 for Repairof Noncritical Size Defects - Unrelated Controls MECHANICAL TESTINGRESULTS Percent Energy absorbed Time Maximum Load to Failure Torqueintact control to failure Animal Number Side Period Implant (N) (Nm) (%)Angulation (Nm-degrees) 17932 right 8 weeks Unrelated Control 35.40 2.1254.24 61.58 34.63 18926 right 8 weeks Unrelated Control 6.20 0.37 9.5059.65 6.96 18754 right 8 weeks Unrelated Control 25.68 1.54 39.34 33.5327.87 17932 left 8 weeks Unrelated Control 29.14 1.54 39.34 33.53 27.8718926 left 8 weeks Unrelated Control 5.67 0.34 8.69 47.20 6.20 18754left 8 weeks Unrelated Control 12.23 0.73 18.74 39.43 15.10 MEAN 19.051.14 29.29 44.64 18.43 STANDARD DEVIATION 12.68 0.76 19.42 14.14 11.36SAMPLE SIZE 6 6 6 6 6

C. Repair of Fracture Defects Using Improved Osteogenic DevicesContaining Carboxymethylcellulose

1. Experiment 1 Goat Fracture Study Using Varying Doses of OP-1 (ClosedDefect Site)

The following is a comparative randomized experimental study of freshclosed tibial midshaft fracture defects (distracted to 5 mm) in goats.

Choice of Experimental Animal

It is generally recognized in the art that goats have a bone healingrate comparable to that of humans. Thus, the results of this study canbe extrapolated to a clinical setting. Moreover, it is appreciated bythe skilled artisan that the bones of goats show similarity to those ofhumans regarding size, shape and mechanical load.

As disclosed and described herein, an animal model for a closeddiaphyseal fracture has been developed. This model promotes the study ofnatural and accelerated fracture healing, with or without an internalfracture fixation device, by permitting creation of a reproduciblestandard fracture of the hind limb. Briefly, in fully anesthetizedgoats, a closed fracture of the midshaft of the tibia is created withthe aid of a three point bending device. After closed reduction anddistraction to 5 mm of the fracture, an external cast is applied.Because of a decrease in the swelling of the hind limb, the cast isreplaced biweekly to retain stability. After 2 weeks, the animals arefull weight bearing on the fractured limb, and after 4–6 weeks thefracture is healed clinically and radiographically. The cast is removedafter 6 weeks.

The animals are purchased from Ruiter (Netherlands), a goat breedingspecialist. Random bred adult female milk-goats will be used. Tocircumvent the influence of a developing skeleton on the results, adultanimals will be used. The animals are skeletally mature, 1 to 2 yearsold and weigh about 50 kg.

Experimental Procedure

As a premedication, ketamin 10 mg/kg i.m. and atropine 1.5 mg i.m. (orart-recognized equivalents of the foregoing medications) areadministered about 15 minutes before fully anesthetizing the animals.The latter is accomplished with etomidates (or art-recognizedequivalents thereof) 0.3 mg/kg i.v. After intubation, anesthesia ismaintained with an O₂/N₂O-mixture (1:1, vol/vol) supplemented with 1 to2% isoflurane (or art-recognized equivalents thereof).

With a 3-point bending device, a varus trauma is applied to the lefttibia until a closed midshaft fracture is obtained. The fracture is thenreduced manually, and the skin over the fracture area is shaved. Thewhole left hind limb is iodinated with an alcohol containingdisinfectant solution for closed osteogenic device administration byinjection and to dry the skin for subsequent cast immobilization. Theosteogenic device is injected at the fracture site in the vicinity ofthe fracture gap to maximize contact with the medullary cavity. Forexample, an osteogenic device is injected intramedullary with a thickbone marrow aspiration needle. After the injection, cast immobilizationis applied.

Study Design

The animals are divided into 5 groups (I–V) of 3 animals and 1 group(VI) of 9 animals according to treatment: 0.5 mg OP-1 in an injectableconfiguration of osteogenic device containing at least OP-1, collagenmatrix, and binding agent such as CMC, formulated as described above(directly after creation of the fracture) (Group I), 1.0 mg OP-1 in aninjectable device containing at least OP-1, collagen, and binding agent,such as CMC, (directly after creation of the fracture) (Group IV), 1.0mg OP-1 in a standard configuration of OP-1 device (corresponding to 0.4gram OP-1 device) injected directly after creation of the fracture(Group V), and no treatment with OP-1 (Group VI, controls). Thetreatment groups are summarized as follows:

Time of Approximate injection Amount of Number of Group (days) DeviceOP-1 (mg) animals I 0 Injectable 0.5 3 II 0 Injectable 1.0 3 III 3Injectable 1.0 3 IV 0 Injectable 1.0 3 V 0 Standard Device 1.0 3 VI NoneNone 0 9

The animals are sacrificed 2, 4 and 6 weeks after creation of thefracture. In groups I to V, one animal is sacrificed at each timeinterval, and in group VI, three animals are sacrificed at each timeinterval. By comparing the treated groups to the controls, theaccelerating effect of treatment on fracture healing can be determined.Information about the OP-1 dose effect and the time of injection can beobtained by comparison of group I to group II, respectively, and groupII to group III. Differences in efficacy between differentconfigurations are assessed by evaluating the results of groups II, IVand V.

In other related experiments, doses of osteogenic protein such as OP-1will range from approximately 0.125 to 10.0 mg. Certain otherconfigurations of improved osteogenic devices will contain varyingamounts of binding agent such as CMC, ranging from below 200 mg CMC/1000mg collagen matrix to above 200 mg CMC/1000 mg collagen matrix. Wettingagent volumes will be varied as earlier described to achieve the desiredconsistency/configuration of osteogenic device.

Evaluating Defect Repair Radiography

X-rays are made following a standardized procedure and depict thefracture site in two directions, anteroposterior and mediolateral. Thefirst radiographs are taken immediately after creation of the fractureand thereafter biweekly until sacrifice of the animals. The radiographsat the time of sacrifice are made after removal of the casting material;all others are made with the casting material in situ. They are judgedqualitatively by two blinded radiologists or surgeons, and, if possible,the following grading scale for evaluating the healing process isapplied:

-   -   Grade 0: No difference compared to directly after creation of        the fracture    -   Grade 1: Small amount of callus    -   Grade 2: Moderate amount of callus    -   Grade 3: Large amount of callus    -   Grade 4: Fading of the fracture ends        Special attention is paid to the type of fracture and alignment.

Computed Tomography

After removal of the left hindlimb and casting material, and aftermaking of the radiographs, a CT-scan of the fracture area is made. Thesoft tissues should remain in situ for a better quality of scans.Remnants of the fracture gap and callus can be made visible in this way.Moreover, the amount of callus can be calculated. More detailedinformation about the progress of the healing process can be obtainedwith CT scans than with plain radiographs.

Biomechanical Test

After CT scanning and subsequent removal of all soft tissues from thetibia, biomechanical investigations are performed. A method for advancedmechanical testing of bone is developed as follows: the bendingstiffness in 24 directions at angular increments of 15° is measured anddepicted as a vector in a X-Y coordinates system, by which an ellipse isobtained. The ellipse is compared with that of the contralateral intacttibia. Parameters can be derived from this comparison that serve asmeasures of the healing efficiency. Finally, a torsion-test-to-failureis done and the measured torsion strength, torsion stiffness, angulardisplacement and energy absorption-to-failure is expressed as apercentage of the contralateral healthy tibia. This comparison with thecontralateral tibia is made to reduce the interindividual variation.

Histology

After biomechanical testing, the bone fragments are held together withspecial rings for histologic examination. Standard fixation, imbeddingand staining techniques for bone and cartilage are used. Specialattention is paid to signs of fibrous, osteochondral or bony union. Ahistologic scoring system is applied to quantitate the amount of fibroustissue, cartilage, newly formed bone and bone marrow in the fracturegap.

Experimental Results

It is expected that mechanical, radiographic, tomographic andhistological data will indicate that injectable configurations ofimproved osteogenic devices can induce accelerated repair of closed sitefracture defects.

Conclusion

CMC-containing osteogenic devices (injectable configuration) can be usedto repair fresh tibial midshaft fracture defects (distracted to 5 mm) ata closed defect site.

2. Experiment 2

Goat Fracture Study Using Varying Doses of OP-1 at Varying Times (ClosedDefect Site)

This independent study also uses goats as the animal model for studyingrepair of fracture defects using improved osteogenic devices. Usingtechniques similar to those described above, fresh closed diaphysealfractures (mostly transverse and simple oblique) with reduction withexternal fixation and distraction to 5 mm are treated usingCMC-containing osteogenic devices.

The study design is as follows:

Group Treatment No. Goats I No injection 10 II CMC + collagen alone viainjection 10 III CMC + collagen + OP-1 (2.5 mg 10 OP-1/1000 mg collagen)via injection IV CMC + collagen + OP-1 (half-maximal 10 dosage of 1.25mg OP-1/1000 mg collagen) via injectionFive goats in each group are sacrificed at 2 weeks post-treatment andfive goats in each group are sacrificed at 4 weeks post-treatment.

Other related studies investigate repair of fracture defects at timepoints greater than 4 weeks, and investigate both lower and higherdosages of OP-1. Additionally, repair of fracture defects usingdiffering total amounts (mg) of the CMC-containing OP-1 deviceadministered at the defect site are studied. One study utilizes a deviceof 400 mg total weight administered at the defect site.

Defect repair is evaluated using a variety of routine clinicalprotocols, including radiography, CT scan, biomechanical testing, andhistology, as described in more detail above.

Experimental Results

It is expected that mechanical, radiographic, tomographical,histological data will indicate that injectable configurations ofimproved osteogenic devices can induce accelerated repair of closed sitefracture defects. It is also anticipated that low doses of osteogenicprotein will be effective to induce repair, especially in improvedosteogenic devices.

Conclusion

CMC-containing osteogenic devices (injectable configuration) can be usedto repair fresh closed diaphyseal fractures (distracted to 5 mm) at aclosed defect site.

D. Repair of Osteochondral Defects Using Improved Osteogenic DevicesContaining Carboxymethylcellulose

1. Experiment 1

Full-Thickness Osteochondral Defects (Dogs)

A study using the dog osteochondral plug defect model was conducted todemonstrate the efficacy of improved osteogenic devices for repairingosteochondral/chondral defects. Four formulations of implants wereevaluated, including (1) standard osteogenic device, including rhOP-1and collagen matrix, (2) improved osteogenic device, including rhOP-1,collagen matrix and carboxymethylcellulose (CMC) binding agent, (3)collagen matrix only, or (4) collagen matrix and CMC binding agent.

Briefly, full thickness defects 5 mm in diameter and extending 6 mm intothe subchondral bone were created bilaterally on the medial femoralcondyle of 4 adult mongrel dogs. Adult male mongrel dogs were chosenbecause of their anatomical size and bone repair and remodelingcharacteristics. Special attention was paid in selecting animals ofuniform size and weight to limit the variability in bone geometry andjoint loading. The animals were radiographically screenedpre-operatively to ensure proper size, skeletal maturity, and that noobvious osseous abnormalities existed. The left side defects receivedstandard osteogenic device in two animals, and the improved osteogenicdevice in the other two animals. The right side defects received matrixalone in one animal, a matrix/binding agent mixture in one animal, andwas untreated in the remaining two animals.

Test Device Description

The standard osteogenic device consisted of rhOP-1 admixed with bovineType I bone collagen matrix (2.5 mg rhOP-1/g matrix). The improvedosteogenic device comprised 100 mg of the OP-1/collagen matrix standardosteogenic device combined with 20 mg of CMC (total of 120 mg). Controlsconsisted of bovine Type I bone collagen matrix alone, and the collagenmatrix with CMC. Both were supplied in 100 mg quantities.

Study Design

Study design is summarized in Table 12.

TABLE 12 Dog Osteochondral Defect Repair using OP-1 Animal Left RightNumber Implant Implant H122 OP-1 Matrix H130 OP-1 None H125 OP-1/CMCNone H132 OP-1/CMC None OP-1: 100 mg OP-1/Collagen Device (standardosteogenic device). OP-1/CMC: 120 mg OP-1/CMC/Collagen Device (improvedosteogenic device). Matrix: 100 mg Collagen. CMC/Matrix: 100 mgCMC/Collagen. Devices and controls were wetted with saline (approx. 0.21to 0.26 ml) to achieve a putty consistency prior to implantation.

Surgery

Using standard aseptic techniques, surgery was performed underisofluorane gas anesthesia. Anesthesia was administered by intravenousinjection of sodium pentothal at a dosage of 5.0 mg/lb body weight. Amedial parapatellar incision approximately four centimeters in lengthwas made. The patella was retracted laterally to expose the femoralcondyle. A 5 mm drill bit with a specially designed sleeve to preventover drilling of the defect depth (6 mm) was used to create the finaldefect. Sterile saline was added to the improved osteogenic device andmixed just prior to implantation. After irrigation of the defect withsaline to remove bone debris and spilled marrow cells, the appropriatedevice was packed into the defect site using a blunt probe. Enoughdevice was placed within the defect so that it was flush with thearticulating surface. The joint capsule and soft-tissues were thenclosed in layers. The procedure was repeated on the contralateral sidewith the appropriate implant.

Evaluations and Terminal Procedures

Osteochondral healing was evaluated grossly and histologically usingroutine protocols, as described below. Radiographs were utilized toevaluate healing.

At twelve weeks post-operative each animal was sacrificed by anintravenous barbiturate overdose. Both right and left distal femurs wereharvested en bloc and kept in cool saline until gross grading andmicrophotography were completed. The specimens were then placed in 4%paraformaldehyde fixative, labeled with all necessary identifications,and stored at 4° C. until shipped approximately 10 days post-sacrifice.Just prior to shipping the specimens were trimmed into small blocks,with the articular defect in the center.

Gross Analysis

Each harvested defect was graded for gross appearance. This analysisapportions points based upon the formation of intra-articular adhesions,restoration of articular surface, erosion and appearance of thecartilage. A total of eight points is possible. The gross grading scaleis set forth in Table 13.

TABLE 13 Gross Grading Scale Grades Intra-articular adhesions None = 2Minimal/fine loose fibrous tissue = 1 Major/dense fibrous tissue = 0Restoration of articular surface Complete = 2 Partial = 1 None = 0Erosion of cartilage None = 2 Defectsite/siteborder = 1 Defect site andadjacent normal cartilage = 0 Appearance of cartilage Translucent = 2Opaque = 1 Discolored or irregular = 0 TOTAL SCORE 8 possible points

Histology

All specimens were prepared for histologic evaluation. The individualspecimens were fixed by immersion in 4% paraformaldehyde solution. Inaddition, using routine procedures as described elsewhere herein, tissuetyping analysis was performed in order to characterize the collagen typeand percent tissue composition. Non-decalcified sections, one from eachspecimen, stained with Safranin-O and Fast Green stains (to indicateglycosaminoglycan content in the matrix), were returned for evaluation.

Histologic sections were based upon the nature of the repair cartilage,structural characteristics, and cellular changes. The histologic gradingscale is set forth in Table 14.

TABLE 14 Histology Grading Scale Nature of the Predominant TissueCellular morphology Hyaline articular cartilage = 4 Incompletelydifferentiated = 2 Fibrous tissue or bone = 0 Safranin-O staining of thematrix Normal/near normal = 3 Moderate = 2 Slight 1 None = 0 STRUCTURALCHARACTERISTICS Surface regularity Smooth/intact = 3 Superficialhorizontal lamination = 2 Fissures, 25–100% of thickness = 0 Severedisruption, fibrillation = 0 Structural integrity Normal = 2 Slightdisruption, including cysts = 1 Severe disintegration = 0 Thickness 100%of normal cartilage thickness = 2 50–100% = 1 1–50% = 0 Bonding to theadjacent cartilage Bonded at both ends of the defect = 2 Bonded at oneend or partially bonded at both ends = 1 1 Not bonded = 0 FREEDOM FROMCELLULAR CHANGES OF DEGENERATION Hypocellularity None = 3 Slight = 2Moderate = 1 Severe = 0 Chondrocyte clustering None = 2 <25% of cells =1 >25% of cells = 0 Freedom from degenerative changes in adjacentcartilage Normal cellularity, no clusters, normal staining = 3 Normalcellularity, mild clusters, moderate staining = 2 Mild or moderatehypocellularity, slight staining = 1 Severe hypocellularity, poor or nostaining = 0 TOTAL 24 possible points

RESULTS

All surgeries were uneventful with no post-operative complications. Ingeneral, some medial knee swelling was observed on post-operative dayfour bilaterally in all four animals and subsided by post-operative dayten. No animal experienced any adverse reaction related to the implantedmaterials or experimental procedures.

Gross Evaluation

A summary of the mean gross evaluation grades appears in Table 15.

TABLE 15 Mean Gross Evaluation Grade ± standard deviation (n) StandardImproved Osteogenic Osteogenic Collagen Collagen No Device Device MatrixOnly Matrix/CMC Treatment Intra- 2.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.02.0 ± 0.0 Articular Restoration 1.5 ± 0.6 1.5 ± 0.6 0.0 ± 0.0 0.5 ± 0.62.0 ± 0.0 of Surface Erosion 1.8 ± 0.5 1.23 ± 0.5 1.5 ± 0.7 1.0 ± 1.41.8 ± 0.5 Appearance 0.8 ± 0.5 1.0 ± 0.8 0.0 ± 0.0 1.0 ± 0.0 1.5 ± 0.6Total 6.0 ± 1.4 (2) 5.8 ± 1.7 (2) 3.5 ± 0.7 (1) 4.5 ± 0.7 (1) 7.3 ± 0.5(2) (out of 8 possible points)

Histological Evaluation

The non-treated defects and defects treated with the improved osteogenicdevice of OP-1, collagen matrix and CMC received the greatest meanhistologic grade, 15 and 16.5 out 24 possible points, respectively. Ineach of these groups, however, one specimen looked markedly better thanthe other. The collagen matrix only, collagen matrix with CMC, and thestandard osteogenic device treated sites however, scored slightly moreconsistently, and lower than, the sites treated with improved osteogenicdevice (n≦2). A summary of the mean histological grades appears in Table16.

TABLE 16 Mean Histologic Evaluation Grade ± standard deviation (n)Standard Improved Collagen Osteogenic Osteogenic Matrix Collagen DeviceDevice Only Matrix/CMC Non Implanted Nature of the 3.5 ± 0.7 4.5 ± 2.11.0 2.0 4.0 ± 2.8 Predominant (2) (2) (1) (1) (2) Tissue Structural 1.5± 0.1 5.5 ± 0.6 7.0 6.0 5.0 ± 2.8 Characteristics (2) (2) (1) (1) (2)Freedom from 4.5 ± 0.7 6.5 ± 2.1 3.0 4.0 6.0 ± 1.4 Cellular (2) (2) (1)(1) (2) Changes of Dengeneration Total 12.5 ± 0.7  16.5 ± 7.8  11.0 12.0  1.5 ± 7.1 (out of 24 (2) (2) (1) (1) (2) possible points)

Unexpectedly, sites treated with the improved osteogenic device achievedthe highest mean scores for the nature of the new repair tissue, for thestructural characteristics of the repair, and for minimizing thedegeneration of the repair cartilage or the surrounding intactcartilage. The improved osteogenic device sites also received thehighest overall total score. These results were weighted by the score ofone animal, in which the cellular and tissue morphology was consistentwith articular cartilage. The repair cartilage was continuous with theintact cartilage and the thickness of the repair was the same as theintact cartilage. The subchondral bone layer was also completelyrestored. Healing was not as advanced in the other sites treated withthe OP-1/collagen matrix with or without CMC. Lower scores were theresult of incomplete differentiation of the repair tissue, incompletesubchondral bone restoration, and uneven thickness of the repair.Residual implant or carrier material was not observed in any section.

Comparisons within animals demonstrated that, in three animals, thedefects receiving devices containing OP-1, with or without CMC (all leftdefects), achieved histologic grades equal to or greater than thecontralateral defect receiving the control matrix or no treatment.

Unexpectedly, the OP-1 device without CMC induced bone and cartilageformation, but in a more disorganized fashion with considerable fibroustissue present. Untreated or carrier alone samples were filled byfibrous cartilage and dense connective tissue.

These data suggest that the unexpected superior repair achieved withimproved osteogenic device is associated with the differences in itsconsistency relative to that of the standard osteogenic device withoutbinding agent, which in turn affects the containment of the device perse at the defect site. Formulation adhesion and disintegrationproperties are expected to be critical in articular cartilage defectsgiven the dynamic nature of the joint.

Immunostaining of Type I and Type II Collagen and Polarized LightMicroscopy

This study also stained sections to compare collagen repair at defectsites treated with: no device, two types of matrix only compositions(matrix and matrix/binding agent), or both matrix compositions withOP-1.

In general, using the collagen Type I antibody, staining of the existingunderlying subchondral bone, as well as the newly regenerated bone, wasobserved. The newly regenerated bone differed slightly from the existingbone by the presence of regions of more disorganized matrix when viewedunder phase contrast microscopy. Using the Type II collagen antibody,the existing articular cartilage stained qualitatively as well as thereparative tissue in the defects, although staining of the new tissuewas less intense. In at least one defect treated with improvedosteogenic device, complete regeneration of the subchondral bone wasobserved with articular-like cartilage regenerated along the top. Thecellular matrix of this regenerated cartilage was not identical to theexisting articular cartilage, but a visible cellular matrix composed oflarge loose bundles could be seen under phase contrast.

Defects Treated with Improved Osteogenic Device. In defects treated withimproved osteogenic device, at least one animal evidenced repair ofarticular cartilage at a macroscopic level. The subchondral bone wasregenerated and a new cartilage layer of near normal thickness was seenby histological staining with toluidine blue and Safarnin O. Theselayers and tissues stained appropriately, with Type I antibody localizedin the subchondral bone and Type II collagen localized in the newcartilage-like layer. There was also some evidence of the regenerationof a zone of calcified cartilage and distinct tidemark in theregenerated cartilage. However, some differences were seen between thenew and existing articular cartilage layer. The new cartilage had ahigher density of chondrocytes and contained loose, disorganized bundlesof fibers visible by phase contrast microscopy or with polarized light.It should be noted that only a single time point during the repairprocess is represented here and that the results of longer or shorterperiods is unknown.

Defects Treated with Standard Osteogenic Device. Defects treated withstandard osteogenic devices showed approximately 50% of the bone wasregenerated in the defect site with in-growth of articular cartilagefrom the edges of the defect. There appeared to be some additional areasof articular cartilage formation next to the newly regenerated bone,with the remainder of the defect filled with reparative tissue. Thereparative tissue stained lightly with collagen Type II, and not withType I collagen, antibodies. More chondrocytes were present with largeloose bundles of matrix surrounding the cells. Treatment with thestandard osteogenic device differed from treatment of the improvedosteogenic device in that the subchondral bone failed to regenerate toits normal level, and dense disorganized fibrous tissue appeared abovethe new cartilage, which caused the top of the defect to bulge with anirregular surface. This fibrous tissue appeared to have morefibroblast-like cells with fibrous bundles arranged parallel to thearticular surface.

Defects Treated with Matrix/Binding Agent. A defect with onlymatrix/binding agent without OP-1 showed regeneration of about one-thirdof the removed subchondral bone, with the remainder filled with areparative tissue This regenerated tissue stained lightly with Type Icollagen antibodies, especially near the bottom of the defect, andshowed stronger staining with the Type II collagen antibody, withstrongest staining near the surface. A dense disorganized visible matrixis apparent in the top half of the reparative tissue, and a moreorganized horizontal pattern of fibers appears in the bottom half.Toluidine blue did not stain the reparative tissue, whereas Safranin Ostained the top and bottom half differentially. The half near thearticular surface stained lightly with Safranin O, and the bottomstained with Fast Green. A similar distinction was observed between thetwo halves of the reparative tissue when stained with Masson Trichrom.Although the reparative tissue did not look like articular cartilage,the region near the articular surface did appear to contain Type IIcollagen, an acidic matrix with perhaps some mucopolysaccharides. Thebottom half had more Type I collagen with less carbohydrate and may bemore connective tissue-like in nature.

The single defect treated with collagen matrix alone did not show anyregeneration of the subchondral bone. The reparative tissue that filledthe defect stained lightly with both collagen Type I and II antibodies.This tissue had an increased fibrous matrix with fibroblastic like cellsand appeared in some areas to be similar to fibrocartilage. This samplewas similar to the treatment with the CMC/collagen matrix alone, withslight localization of both Type I and II collagen in the reparativetissue. In addition, the defect site showed the same differentialstaining with Safranin O/Fast Green, with staining of the top half ofthe reparative tissue with Safranin O and the bottom with Fast Green.

Summary and Conclusion

Osteochondral defects treated with the improved osteogenic devicesunexpectedly demonstrated more advanced cartilage regeneration,chondrocyte and cartilage phenotype compared to defects treated with thestandard osteogenic device, collagen matrix alone, or collagen matrixadmixed with CMC, all of which demonstrated less organized repaircartilage and subchondral bone formation. Poor repair by treatment withthe collagen matrix or collagen matrix with CMC indicates that thepresence of a collagen scaffold alone is not sufficient to inducehealing and may actually deter the progression of healing andorganization of repair tissue.

Full-thickness osteochondral defects can be repaired usingCMC-containing osteogenic devices in accordance with the methods of theinstant invention.

2. Experiment 2

Long Term Evaluation of Repair of Full-Thickness Osteochondral Defects(Dogs)

This study was conducted to further evaluate repair ofosteochondral/chondral defects by improved osteogenic devices. To date,the study examined the effects of the improved osteogenic device at 6and 12 weeks and will continue to examine effects at 26 and 52 weeks.This provides long term repair stability data. The organization of newcartilage over time was followed to determine if it approximates normaltissue with respect to its structure and function. Two formulations ofdevices were evaluated in osteochondral/chondral defects including: 1)improved osteogenic device, or 2) mock devices containing CMC andcollagen matrix only.

Briefly, full thickness defects 5 mm in diameter extending 6 mm into thesubchondral bone were created bilaterally on the medial femoral condyleof 16 adult mongrel dogs. Adult mongrels were utilized in this studybecause of their anatomical size and known bone repair and remodelingcharacteristics. All animals were between 1 and 4 years old and weighapproximately 20 to 30 kg. Specific attention was paid to selectinganimals of uniform size and weight to limit the variability in jointloading. The animals were radiographically screened to ensure propersize, skeletal maturity, and that no obvious osseous abnormalitiesexist. In each group of four dogs, the left side defects receivedimproved osteogenic device. The right side defects receivedmatrix/binding agent in two animals, and the remaining two animals wereuntreated. At sacrifice, the distal femurs were retrieved en bloc, andthe defect sites evaluated histologically and grossly based on upon theabove-described scheme.

The improved osteogenic device comprises standard device (2.5 mgrhOP-1/1g matrix) admixed with CMC. To formulate the improved device,100 mg of the rhOP-1/collagen mixture were admixed with 20 mg of CMCimmediately prior to implantation (total 120 mg). The collagen onlydevice consists of bovine Type I collagen (100 mg). The study design issummarized in Table 17.

TABLE 17 Dog Osteochondral Defect Repair Dogs Group (2 defects/animal)Left Implant Right Implant Duration I 4 OP-1/CMC None/Vehicle  6 weeksII 4 OP-1/CMC None/Vehicle 12 weeks III 4 OP-1/CMC None/Vehicle 26 weeksIV 4 OP-1/CMC None/Vehicle 52 weeks OP 1/CMC: 120 mg OP-1 CMC/CollagenDevice (improved osteogenic device) Vehicle: 100 mg CMC/Collagen.

Surgery

Using standard aseptic techniques, surgery was performed underisofluorane gas anesthesia. A medial parapatellar incision approximatelyfour centimeters in length was made. The patella was retracted laterallyto expose the femoral condyle. Using a ⅛ inch drill bit, a pilot holewas made in the weight bearing region of the medial femoral condyle. A 5mm drill bit with a specially designed sleeve to prevent over drillingof the defect depth (6 mm) was used to create the final defect. Aftercopious irrigation with saline to remove bone debris and spilled marrowcells, the appropriate experimental device was packed into the defectsite using a blunt probe. The joint capsule and soft-tissues were thenmeticulously closed in layers. The procedure was repeated on thecontralateral side with the appropriate implant.

Evaluation

Four animals each were sacrificed at 6 and 12 weeks and four animalswill be sacrificed at 26 and 53 weeks post-operative. Animals weresacrificed using an intravenous barbiturate overdose. The femurs wereimmediately harvested en bloc and stored in a saline soaked diaper. Highpower photographs of the defect sites were taken. Soft-tissues weremeticulously dissected away from the defect site. The proximal end ofthe femur was removed.

The gross appearance of the defect sites and repair tissue were gradedbased upon the above-described parameters by two independent observersblinded to the treatment assignment. Points were apportioned accordingto the presence of intra-articular adhesions, restoration of thearticular surface, cartilage erosion and appearance.

All specimens were prepared for histologic evaluation immediately aftergross grading and photography. The individual distal femurs were fixedby immersion in 10% buffered formalin solution or in 4% paraformaldehydesolution. On a water cooled diamond saw, each defect site was isolated.Three sections from three levels were cut from each block. Levels 1 and3 were closest to the defect perimeter. Level 2 was located at thedefect center. Three sections from each level were stained with eitherhematoxylin and eosin, Goldner's trichrome, Safranin O, or Fast Green.Sections were then graded based upon the above-described scheme. Thisanalysis apportioned points based upon the nature of the repair tissue,structural characteristics, and cellular changes. A total of 24 pointsare possible.

Result and Conclusion

After 6 weeks, certain of the above-treated animals were sacrificed andimmunohistochemical evaluations were conducted as described elsewhereherein. The results were as follows: In all cases, defects treated withOP-1 CMC/collagen device exhibited superior repair. With the OP-1CMC/collagen device, there was unexpectedly complete or nearly completebridging of the defect with cartilage tissue. Type II collagen stainingwas observed in the reparative cartilage with little or no Type Icollagen staining. Proteoglycan staining followed the type II collagenlocalization with darker staining in areas that more closely resembledmature hyaline cartilage. Based on Safranin-O staining, regeneration ofsurface layer of cartilage was not yet complete at 6 weekspost-treatment.

After 12 weeks, healing had significantly progressed in defects treatedwith improved devices. No appreciable healing was observed in thecontrols. The mean gross grading score observed with improved devices at12 weeks was 6.50±0.89 (n=8); control means was 3.69±0.70 (n=8). At allremaining time points, it is anticipated that defects treated with theimproved osteogenic devices will demonstrate more advanced cartilageregeneration, chondrocyte and cartilage phenotype, in an acceleratedmanner relative to defects treated with only collagen/CMC or leftuntreated. The defects treated with improved osteogenic device areanticipated to exhibit cartilage and subchondral bone tissue, whereasthe collagen/CMC treated or untreated defects are expected to inducedisorganized bone and cartilage formation with considerable fibroustissue present.

Full-thickness osteochondral defects can be stably repaired usingCMC-containing osteogenic devices in accordance with the methods of theinstant invention.

E. Repair of Chondral Defects Using Improved Osteogenic DevicesContaining Carboxymethylcellulose

1. Experiment 1

Long Term Evaluation of Repair Chondral vs. Osteochondral Defects(Sheep)

This study evaluates repair of both chondral and osteochondral defectsby improved osteogenic devices using a large animal model. The increasedthickness of the articular cartilage and the similarities to humans insize and weight-bearing characteristics make the sheep a model fromwhich human clinical applications can be extrapolated, especially forclinical application of improved osteogenic devices for repair ofchondral defects. The study groups are as follows:

A-Osteochondral (full-thickness) Defects (5 mm diameter) Group A I: notreatment Group A II: carboxymethylcellullose/collagen Group A III:carboxymethylcellullose/OP-1/collagen Group A IV: lyophilized allograftGroup A V: lyophilized allograft + OP-1 B-Chondral (partial thickness)defects (5 mm diameter) Group B I: no treatment Group B II:carboxymethylcellullose/collagen Group B III:carboxymethylcellullose/OP-1/collagen Group B IV: hyaluronic acid +chondroitin sulfate paste Group B V: hyaluronic acid + chondroitinsulfate paste + OP-1

Both foreknee joints of each sheep are operated on, and two defects perjoint are created (one each on the medial and the lateral condyle). Oneof the joints has two standardized partial thickness chondral defects (5mm in diameter) created on each condyle, while the other joint has twodeeper, full thickness osteochondral defects (about 1–2 mm into thesubchondral bone) created. Each group has a subgroup sacrificed early at8 weeks and another kept for longer term evaluation for 6–7 months.

There are a total of 20 groups and 12 defects per group. Therefore, thetotal number of defects is 240 and total number of sheep is 60. Thereare five different treatment groups; three controls (no treatment andtwo different mock devices) and two different OP-1 formulations for eachdefect type. Improved osteogenic devices comprising OP-1 in CMC/collagenwill be used for osteochondral defect repair and chondral defect repair.The devices are formulated such that 2.5 mg OP-1/g collagen are added toeach defect site receiving this improved osteogenic device. Repair isevaluated at 8 weeks and 6–7 months. The treatment protocol is shown inTable 18.

TABLE 18 Sheep Chondral and Osteochondral Defect Repair using OP-1 SheepOsteochondral Chondral Defects Group (4 defects/sheep) (2 defects/sheep)(2 defects/sheep) Duration I 12 Untreated Control Untreated Control  8weeks (6) >26 weeks (6) II 12 CMC/Collagen CMC/Collagen  8 weeks (6)Control Control >26 weeks (6 III 12 OP-1 + OP-1 +  8 weeks (6)CMC/Collagen CMC/Collagen >26 weeks (6

Surgeries on the two knees are staggered by two weeks to allow healingof the first knee prior to surgery on the second knee. The first surgeryis used to generate chondral defects, the second is for osteochondraldefects. The surgery is performed in a fully equipped operating roomusing standard techniques and equipment used in human surgery. The sheepare allowed to ambulate freely in their pasture territorypost-operatively. Staggered surgeries result in 8 week healing times forchondral defects and 6 week healing times for osteochondral defects. Atsacrifice, the joints are perfused, fixed and processed according tostandard cytological protocols.

At the end of the study periods, the animals are sacrificed and thejoints are harvested en bloc. The gross appearance of the defect sitesand repair tissue is graded using routine methods such as thosedescribed above. Points are apportioned according to the presence ofintra-articular adhesions, restoration of articular surface, cartilageerosion and appearance.

Using methods similar to those described above, specimens are preparedfor histologic evaluation immediately after gross grading andphotography.

It is expected that defects treated with OP-1/CMC/collagen devices willexhibit superior repair similar to theat in Experiment D.2 above.

2. Experiment 2

Long Term Evaluation of Repair of Using Varying Doses of OP-1Subchondral Defects (Goats)

A study using skeletally mature milk-goats is conducted to demonstratethe efficacy of improved osteogenic device for repairingosteochondral/chondral defects. Formulations of improved osteogenicdevice with varying concentrations of rhOP-1 are used, along with mockor no-device controls. The mock device consists of collagen admixed withcarboxymethylcellulose (CMC). Furthermore, the animal groups aresacrificed at 4, 12 and 24 months after surgery to compare the rate andstability of defect repair. The following summarizes the experimentalparameters.

Groups Post-operation time: 4 mo. 12 mo. 2 year 1. rhOP-1 800 μg/ml A BC 2. rhOP-1 1600 μg/ml A 3. rhOP-1 3200 μg/ml A 4. Mock device A B 5. Nodevice A B

Briefly, subchondral defects are made in the left knees of 56 skeletallymature milk-goats: The defects are 8 mm in diameter and 3 mm in depth.This defect configuration prevents very high shear stresses in thedefect leading to collagen Type I formation. Dutch milk-goats, about 2years old and weighing approximately 50 kg are used in this experiment.Devices corresponding to 2.5 mg rhOP-1/gram collagen are provided. Ineach case, 0.2 grams of CMC are added to standard osteogenic device,then approximately 2.6 ml of saline are added and mixed. This yieldsmaterial of approximately 3–4 ml of improved osteogenic device. Thismaterial is then used to fill the defect volume.

Surgical Technique

Anesthesia is induced and the left knee is opened via a medialparapatellar approach. The patella is dislocated to the lateral side andthe medial condyle is exposed. With a sharp hollow tube, the outlines ofa defect are made in the anterior weight bearing part of the medialcondyle. With a square pointed handburr that is placed inside the tube,a defect down to the subchondral bone is created. The proximal tibia isthen exposed, and a periosteal flap of the same diameter as the defectin the medial condyle is taken. The periosteal flap is partially fixed,with its cambium layer towards the defect, to the remnants. The defectis filled with the appropriate test material and covered with theperiosteal flap, using a resorbable suture. The CMC device is added viaa syringe until the defect is filled, and the flap is then completelysutured. In control animals, a mock device including collagen and CMConly is used. A second control group received no implant at all, butreceived only a periosteal flap.

Post-operative treatment

Unrestricted, weight-bearing activity is allowed as much as can betolerated post-operatively.

Clinical performance

The weight bearing pattern is assessed at 2, 4, 6, and 8 weeks, and thenevery 4 weeks.

Gross Analysis

Gross evaluations are made based upon the scheme presented above. Aftersacrificing the animal, the presence or absence of knee contractures isrecorded, and both the patella and condyles of the femur are examinedfor adhesions, articular surface contour, the appearance of the restoredcartilage, and the presence or absence of cartilage erosions. Each ofthese characteristics is given a score. Color slides are taken using amacro-lens.

Histological analysis

To aid in visualization of the regenerated subchondral bone and tolocalize the borders of the defect during histological evaluation, thegoats receive a double labeled tetracycline before sacrificing. Thisallows histomorphometry of the bony filling of the deeper part of thedefect. The histological samples are also viewed by incorporatingpolarized microscopy to provide information on regular structuralfeatures.

For histological analysis, the specimens, including the subchondralbone, are fixed in 10% phosphate buffered formalin and are embeddedundecalcified in methylmethacrylate (MMA). With a heavy duty microtome,sections of 5 μm thick are made. The sections are stained with toluidineblue to identify cartilage and with Goldner's Trichrome to identifybone. Assessment is made of tissue hyalinity, affinity of the matrix fortoluidine blue (metachromasia), surface irregularity, chondrocyteclustering regenerated subchondral bone, bonding to the adjacentarticular cartilage, inflammatory cell infiltration around the implant,and freedom from degenerative changes in the adjacent cartilage. Each ofthese characteristics is given a score.

Biochemical Analysis

Extraction of proteoglycans: For biochemical analysis, control cartilageand tissue from the defect is collected in cold phospate-buffered saline(PBS). Proteoglycans are extracted from lyophilized sections bytreatment with 4 M guanidine HCl, 0.15 M potassium acetate at pH 5.8 inthe presence of proteinase inhibitors (5 mM benzamidine, 0.1 M6-amino-n-hexanoic acid, 10 mM EDTA, 5 mM phenylmethylsulfonyl fluoride,and 5 mM n-ethylmaleimide) at 4° C. for 60 hours. The extract andresidue are separated. The residue is thoroughly rinsed with extractionbuffer, which is added to the extract. The extracts are analyzed forchondroitinsulphate content and used for gel filtration.

Gel filtration: Aliquots of extracts are applied to Sepharose C 12 Bcolumns (0.66×145 cm) (Pharmacia AB, Uppsala, Sweden) and eluted with adissociative buffer at pH 6.1, containing 4 M guanidine HCl, 0.1 sodiumsulfate, 0.05 M sodium acetate, and 0.1% triton X-100. The flow rate is1.2 ml/hr. Fractions are analyzed for chondroitinsulphate content. Theamount of large cartilage-specific molecules, probably aggrecans, can becalculated.

MRI

Magnetic resonance imaging (MRI) is performed for 2 purposes. First, tomonitor 1 month post-operatively that the flap plus implant has remainedin place. Second, group 1C (2 years or more post-op) is followedlongitudinally with MRI at 4 months, 12 months and at sacrifice.

Summary

It is anticipated that defects treated with improved osteogenic devicewill demonstrate advanced cartilage regeneration, chondrocyte andcartilage phenotype compared to the mock or no-device controls. It isalso anticipated that low doses of OP-1 will at least achieve repairquantitatively and qualitatively similar to that of higher doses.

F. Repair of Chondral Defects Using Osteogenic Protein

This study investigated mammalian cartilage formation in subchondrallesions treated with recombinant human osteogenic protein-1 (rhOP-1)(alone or in combination with a collagen matrix) and/or autologousperichondrium.

Material and Methods

In the medial femoral condyle of the left knee joint of 15 goats, asubchondral defect of 9 mm diameter was made. The defect was filled withan implant consisting of fresh coagulated blood mixed with: (a) smallparticles of autologous ear perichondrium; or (b) rhOP-1; or (c) rhOP-1plus ear perichondrium. Rh-OP-1 was either added in combination with acollagen matrix (OP-1 Device) or without a collagen matrix (OP-1 alone).The defect was closed with a periosteal flap, which was stitched to thecartilage. After implantation times of 1, 2 and 4 months, the extent ofrepair of each defect was investigated with standard histologicaltechniques (metachromasie and hyalinity) and well-known biochemicalmethods (gel chromatography of proteoglycans).

Results

After 1 and 2 months in this particular study, there were no apparentdifferences between control (implant (a) above) and the various OP-1treated defects. However, after 4 months, only one out of three controldefects showed detectable cartilage formation, while all four OP-1treated defects were completely or partly filled with cartilage, asindicated by the histological and biochemical analysis set forth inTable 19.

TABLE 19 A B % of Biochemical Histology Cartilage Score² Implant defect¹Score Score partial total Control 86% 0.7 0.0 0.60 14% 3.0 4.0 0.98 1.58OP-1 62% 2.0 4.0 3.72 Device 38% 3.3 6.0 3.53 7.25 OP-1 79% 1.2 2.0 2.53Device + perichon. 21% 5.7 6.0 2.46 7.99 OP-1 79% 2.0 5.0 5.53 alone 21%2.1 6.0 1.70 4.23 OP-1 + 78% 1.0 2.0 2.34 perichon. 22% 4.2 5.0 2.024.36 ¹The defect was divided into homogeneous parts, the % is indicated.²Calculated as follows: % × (A + B), e.g. 0.86 × (0.7 + 0.0) = 0.60.

Table 19 sets forth the cartilage score of condylar defects, treated for4 months without OP-1 (control) or with OP-1 plus or minus perichondriumin the presence or absence of a collagen matrix.

Biochemical score (A) was assigned a value from 0–5 based on gelchromatography.

Histology score (B) is based on undecalcified plastic sections on agrading scale of 0 to 6.

Conclusion

The results of this study confirm that OP-1 has cartilage-promotingutility in large subchondral defects in goats. This indicates that OP-1is of clinical relevance in treating large lesions of articularcartilage and is particularly useful for chondral repair ofweight-bearing skeletal defects caused by trauma or disease in mammals.

In related studies, it is anticipated that other improved osteogenicdevices will result in similar repair of large subchondral defects. Itis further anticipated that subchondral defect repair will occur at anaccelerated rate with reduced amounts of OP-1 when admixed with collagenmatrix and a binding agent, such as CMC, relative to OP-1 admixed withcollagen alone. Moreover, such repair will be accompanied byregeneration of more stable, pristine articular cartilage.

G. Segmental Defect Repair (Critical and Non-critical Size) UsingImproved Osteogenic Devices Comprising Apatites and/or TriCalciumPhosphates (TCP and/or Collagen Matrices

Improved devices comprising a variety of matrices or admixtures thereofwill be used to repair segmental ulna defects (critical and non-criticalsize) at varying doses of OP-1 in rabbits and dogs. Improved deviceswill comprise: Pyrost® matrix (Osteo AG, Switzerland), a HAp blockderived from bovine bone; 100% HAp granules (approximately 300–400 or350–450μ); 100% TCP (approximately 400μ); and 50% HAp/50% TCP(approximately 400μ). Other embodiments will comprise one or more of theearlier-described matrices of appropriate porosity. One particularlypreferred embodiment of improved osteogenic device will compriseCollapat® matrix (Osteo AG, Switzerland), a sponge of HAp and collagen.Another particularly preferred embodiment comprises approximately 0.6 gCMC per g HAp granules or per g granules of 75% HAp/25% TCP, especiallywhen a device with putty consistency is desired.

It is expected that improved devices such as those described above willinduce repair of segmental defects, especially at low doses of OP-1.

VI. Human Clinical Studies: Methods of Use of Improved OsteogenicDevices

A. Repair of Bone Defects.

1. Trial 1: Fresh Open Tibial Fracture

This study is a multi-center, prospective, randomized study of patientswith fresh, fractures of the tibia requiring surgical intervention atthe fracture site.

Introduction

Currently there are approximately 26 million fractures annually worldwide. The majority of fractures heal without complication and are notconsidered “a problem”. There is a “quality of life impact,” however,with patients being out of work or prevented from engaging in normalactivity, not being able to return to activity or, when they do,suffering with lingering pain. Patients, particularly in the westernworld, are growing to expect solutions to these problems.

The cost of fracture treatment is astounding. In 1988, fractures cost anestimated $20 billion in the United States. The largest segment,approximately 44% or $7.2 billion, was related to in-patient treatment.In that year, almost 900,000 persons were hospitalized for fractures,with an average length of stay of 8.8 days for a total of 7.9 milliondays. Nursing home costs were second at $2.8 billion, and out-patienthospital care third at $1.8 billion.

When the economic ramifications of fractures are considered inconjunction with the quality of life impact, there is indeed a need forimprovement in treatment methodologies, especially fractures that areconsidered potentially problematic. These are fractures that because ofthe nature of the injury or mitigating host issues, may requireadditional surgical interventions, take an extended time to heal, and/ormay prevent full functional recovery.

Thus the study described below is designed to investigate improvedosteogenic devices as a healing accelerator for fresh fractures inhumans and as a means to decrease the potential for post-injury problemhealing requirement intervention to augment the healing process.Additionally, certain patients within this study will be treated withimproved osteogenic devices as a bone graft substitute in patientsrequiring bone grafting post-injury or in cases of delayed healing.

As contemplated herein and described above, currently preferredembodiments of improved osteogenic devices have a consistency which canbe injected through a large gauge needle or can be placed through anopen incision such that it will remain generally in place in a bloodyenvironment. In addition to more conventional packaging, the injectableimproved osteogenic devices can be packaged in applicator/syringe readyto be used. A variety of nozzles and needles can be added to customizeapplication. Other embodiments will also be rendered radio opaque byaddition of radio opaque components such as described earlier.

It is expected that the improved osteogenic devices of the instantinvention (injectable and implantable) will decrease the incidence ofadditional interventions, speed rate of healing, improve quality of lifeand speed return to normal activity. Furthermore, it is expected thatthe improved osteogenic devices disclosed herein will be used infractures of all long bones, clavicle, and scapula to promote healing,leading to decreased incidence of intervention (including re-operation),increased speed of healing, increased rate of return to normal activity,and decreased morbidity. In contrast to currently available fracturerepair modalities, there is no biomechanical requirement with theimproved devices and methods disclosed herein.

Study Design

Patients will require surgical treatment of open fractures of the tibiaacquired secondary to trauma. The fracture must have the potential to beadequately stabilized at the fracture site to permit healing. Patientswill show radiographic evidence of skeletal maturity.

Type of Treatment

-   -   Type #1: Initial injury at ≦7 days at definitive closure.    -   Type #2 At up to 6 weeks post-initial injury in patients        requiring bone grafting.

Type #1 fractures are those not requiring bone grafting. Patients willbe randomized in a 1:1 ratio of standard treatment (debridement offracture site, reduction and stabilization), which will be the controlgroup versus standard treatment plus OP-1 device with and without abinding agent, such as carboxymethylcellulose (CMC). In certainpatients, dosages of osteogenic protein OP-1 will vary. As describedabove, a currently preferred formulation of the improved osteogenicdevice contains 2.5 mg OP-1/1000 g collagen/200 mg CMC. OP-1 dosageswill vary from ½ maximal to 4×; CMC content will vary from 100–300 mg.As also described above, variations of wetting agent volumes will beinvestigated by the attending surgeon/physician to achieve the desiredconsistency/configuration of device. Patients from the first group whoare not healed 6 months post-treatment will again be randomized in a 1:1ratio of bone grafting (control) versus OP-1.

Type #2 fractures are those requiring bone grafting. Patients will berandomized in a 1:1 ratio of bone grafting (control) versus OP-1.Patients from the first group who are not healed 6 months post-treatmentwill be crossed over from bone grafting to OP-1 and from OP-1 to bonegrafting.

Study Plan

Patients will be followed for a minimum of 1 year post-treatment toassess healing, with a 24 month follow-up to assess status.

Follow-up assessments will be performed at 2 weeks, 4 weeks and every 4weeks up to 6 months, and at 8, 10 and 12 months post-treatment. Allpatients will have an additional follow-up assessment at 24 months todetermine overall health and fracture site status. The followingassessments will be performed: changes in physical examination;radiographs; clinical pain assessments; clinical assessments ofweight-bearing; clinical assessments of function; quality of lifeassessment (pre-discharge, 6 and 12 months); and documentation of anyinterventions to augment/promote healing (surgical and nonsurgical) andhardware failures/replacements.

It is expected that fractures treated with improved osteogenics willevidence an accelerated rate of healing.

Additionally, it is expected that the patients treated with improvedosteogenic devices will experience at least the following additionalbenefits:

-   -   1) Potential for decreased healing time with faster restoration        of function, weight bearing and ambulation;    -   2) Potential for prevention of delayed/mal/non-union;    -   3) Return to normal activities sooner/less time lost from        jobs/school;    -   4) Potential saving from further intervention/surgical        procedures for promotion of healing;    -   5) Less hardware complications; and    -   6) In those patients who require bond grafting, the benefit of        no second site surgery for bone harvest with associated        morbidity.

2. Trial 2: Fresh Closed Diaphyseal Fracture

Repair of fresh closed diaphyseal fractures in human subjects will beevaluated using improved osteogenic devices. Specifically, patients willbe treated with injectable improved osteogenic devices by injecting thedevice at the closed defect site. It is anticipated that acceleratedrepair of the defect will be observed relative to patients not treatedwith improved osteogenic devices.

B. Repair of Osteochondral Defects.

1. Experiment 1

Osteochondritis Dessicans

Osteochondral defect models support the clinical use of rhOP-1 to treatOsteochondritis Dissecans (OD) and trauma defects. OD is a diseaseresulting in localized areas of osteochondral defects. One cause of thedisease may be ischemia damage to the localized area, but its exactetiology is unknown. In patients with OD, the affected area becomesavascular, with subsequent changes in the overlying articular cartilage.Patient's suffering from OD of the knee experience symptoms includinglocking of the joint, localized pain, swelling and retropatellarcrepitus. An experiment involving patients with OD of the knee isconducted in order to compare the ability of improved osteogenic device,against that of standard osteogenic device, to repair OD defects.

Current methods known in the art for the treatment of OD involve the useof highly invasive surgical techniques. In most skelatally maturepatients with OD, surgery is required. Surgical techniques requirearthroscopic drilling of the intact lesion. As a result, patients mustundergo administration of general anesthesia during surgery.Post-operatively, patients must have movement of their knees restrictedby an immobilizing brace and cannot walk without the use of crutchesuntil healing is evidenced.

In this study, less invasive techniques for treatment of OD areconducted. The techniques involve the use of improved osteogenic device,which is delivered to the defect site via injection. The activity ofimproved osteogenic device in repair of OD is compared to that ofstandard osteogenic device.

It is anticipated that patients treated with the improved osteogenicdevice will show greater relief of symptoms of OD than those treatedwith standard osteogenic device. Patients treated with improvedosteogenic device will experience at least a greater decrease in pain,swelling and locking of the knee than those treated with standardosteogenic device, all of which are indicia of amelioration and/orrepair of the defect.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. A device for inducing local bone or cartilage formation, comprising:a bone morphogenetic protein selected from the group consisting of OP1,OP2, OP3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, BMP10, BMP11, BMP12,BNP15, BMP16, DPP, Vgl, 60A protein, GDF-1, GDF3, GDF5, GDF6, GDF7,GDF8, GDF9, GDF10 and GDF11, capable of inducing repair of endochondralbone, or cartilage, chondral, or osteochondral defects; a matrixselected from the group consisting of collagen, apatites,hydroxyapatites, tricalcium phosphate, and admixtures thereof; and abinding agent selected from the group consisting of cellulosic agentsand salts thereof; wherein said binding agent has a degree ofsubstitution of 0.65–0.90 and a viscosity of about 10–200 cP at a 4%(w/v) concentration of said binding agent.
 2. The device of claim 1,wherein said bone morphogenetic protein is selected from the groupconsisting of OP1, OP2, BMP2, BMP4, BMP5, and BMP6.
 3. The device ofclaim 1 wherein said bone morphogenetic protein is OP-1.
 4. The deviceof claim 1 wherein said device comprises at least two different bonemorphogenetic proteins.
 5. The device of claim 1 wherein said claimmatrix is collagen.
 6. The device of claim 1 wherein said devicecomprises at least two different matrix materials.
 7. The device ofclaim 1 wherein said binding agent is an alkylcellulose.
 8. The deviceof claim 1 wherein said binding agent is selected from the groupconsisting of methylcellulose, methylhydroxyethylcellulose,hydroxyethylcellulose, hydroxypropylmethylcellulose,carboxymethylcellulose, sodium carboxymethylcellulose,hydroxyalkylcelluloses, and admixtures thereof.
 9. The device of claim 1wherein said binding agent is carboxymethylcellulose or a sodium saltthereof.
 10. The device of claim 1 wherein said device comprises atleast two different binding agents.
 11. The device of claim 1 furthercomprising a wetting agent.
 12. The device of claim 1 wherein thebinding agent to matrix ratio is one part by weight binding agent to1–10 parts by weight matrix.
 13. The device of claim 1 wherein thebinding agent to matrix ratio is 1–10 parts by weight binding agent to 1part by weight matrix.
 14. The device of claim 3, wherein the amount ofthe OP-1 ranges from approximately 0.125 mg to 10.0 mg.
 15. The deviceof claim 14, wherein the amount of the OP-1 is approximately 3.5 mg. 16.The device of claim 11 wherein said wetting agent is saline.
 17. Thedevice of claim 12 wherein the binding agent to matrix ratio is one partby weight binding agent to 5 parts by weight matrix.
 18. The device ofclaim 12 wherein the binding agent to matrix ratio is one part by weightbinding agent to 1–5 parts by weight matrix.
 19. The device of claim 13wherein the binding agent to matrix ratio is fewer than 10 parts byweight binding agent to one part by weight matrix.
 20. A device forinducing local bone and cartilage formation comprising a bonemorphogenetic protein comprising an amino acid sequence having at least70% homology with the C-terminal 102–106 amino acids, including theconserved seven cysteine domain, of human OP1; matrix selected from thegroup consisting of collagen, apatites, hydroxyapatites, tricalciumphosphate, and admixtures thereof; and a binding agent selected from thegroup consisting of cellulosic agents and salts thereof; wherein saidbinding agent has a degree of substitution of 0.65–0.90 and a viscosityof about 10–200 cP at a 4% (w/v) concentration of said binding agent;and wherein said bone morphogenetic protein is capable of inducingrepair of endochondral bone when implanted together with a matrix in amammal.
 21. A device for inducing local bone or cartilage formation,comprising at least approximately 1.25 mg of OP-1 and at leastapproximately 180 mg of carboxymethylcellulose per 1000 mg of collagenmatrix, wherein said carboxymethylcellulose has a degree of substitutionof 0.65–0.90 and a viscosity of about 10–200 cP at a 4% (w/v)concentration of said carboxymethylcellulose.
 22. The device of claim 21comprising at least approximately 2.5 mg of OP-1 per 1000 mg of collagenmatrix.
 23. The device of claim 21 or 22 comprising at leastapproximately 200 mg of carboxymethylcellulose per 1000 mg of collagenmatrix.
 24. The device of claim 21 or 22 further comprising saline. 25.The device of claim 23, further comprising saline.
 26. A device forinducing local bone or cartilage formation comprising: OP-1; collagenmatrix; and carboxymethylcellulose having a degree of substitution of0.65–0.90 and a viscosity of about 10–200 cP at a 4% (w/v) concentrationof said carboxymethylcellulose.
 27. A kit for inducing local bone orcartilage formation, the kit comprising: (a) a first receptacle housinga bone morphogenetic protein selected from the group consisting of OP1,OP2,OP3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, BMP10, BMP11, BMP12, BMP15,BMP16, DPP, Vgl, 60A protein, GDF-1, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9,GDF10 and GDF11 and a matrix selected from the group consisting ofcollagen, apatites, hydroxyapatites, tricalcium phosphates andadmixtures thereof, and (b) a second receptacle housing a binding agentselected from the group consisting of cellulosic agents, and saltsthereof, wherein said binding agent has a degree of substitution of0.65–0.90 and a viscosity of about 10–200 cP at a 4% (w/v) concentrationof said binding agent.
 28. The kit of claim 27 further comprising areceptacle adapted to house a wetting agent.
 29. A kit for inducinglocal bone or cartilage formation, the kit comprising: a firstreceptacle housing a bone morphogenetic protein selected from the groupconsisting of OP1, OP2, OP3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, BMP10,BMP11, BMP12, BMP15, BMP16, DPP, Vg1, 60A protein, GDF-1, GDF3, GDF5,GDF6, GDF7, GDF8, GDF9, GDF10 and GDF11, a matrix selected from thegroup consisting of collagen, apatites, hydroxyapatites, tricalciumphosphates, and admixtures thereof, and a binding agent selected fromthe group consisting of cellulosic agents, and salts thereof, whereinsaid binding agent has a degree of substitution of 0.65–0.90 and aviscosity of about 10–200 cP at a 4% (w/v) concentration of said bindingagent.
 30. The kit of claim 29, further comprising a second receptacleadapted to house a wetting agent.